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A farewell to arms The tropicalis mutation xenopus de milo

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					A farewell to arms: The X. tropicalis mutation xenopus de milo abrogates forelimb
formation
Anita Abu-Daya, Satoko Nishimoto, Malcolm Logan and Lyle Zimmerman. National Institute
for Medical Research, Mill Hill, London, UK.

The Xenopus tropicalis insertional mutation xenopus de milo (xdm) was identified in a line
carrying an Nkx2.5-GFP transgene. Homozygous transgenic tadpoles develop normally up
to metamorphosis but do not develop forelimbs, although hindlimb development is
unaffected. Ligation-mediated PCR located the transgene insertion in the third intron of
nephronectin, a ligand for integrin a8b1. Only 20bp of nephronectin intron 3 sequence is
deleted, and SSLP markers on either side of the nephronectin locus are genetically tightly
linked to the transgene, indicating that the transgenesis procedure did not result in major
chromosomal rearrangement or genomic damage. A second transgenic line bearing an
independent Nkx2.5-GFP transgene gave no homozygous phenotype. Nephronectin
messege downstream of the insertion is not expressed in homozygous transgenic tadpoles,
and the xdm phenotype is closely linked to the transgene. Nephronectin morpholino injected
unilaterally into a 2 cell embryo phenocopies the mutation, resulting in one-armed froglets
with low but statistically significant penetrance, confirming the requirement for this gene in
forelimb development in amphibians.

gata transcription factors link canonical and non-canonical Wnt signalling during
cardiogenesis
Boni Afouda and Stefan Hoppler, Institute of Medical Sciences Foresterhill, AB25 2ZD
University of Aberdeen, Aberdeen, Scotland

Wnt signalling has multiple roles during heart development, which involve both inhibition of
cardiogenesis by canonical Wnt/β-catenin signalling and stimulation by non-canonical
Wnt11/JNK signalling. However, little is known about how these two pathways interact to
control the process of cardiogenesis. Here, we use stem-cell-like embryonic explants from
Xenopus laevis to investigate the timing of Wnt function and the molecular basis of the
regulatory mechanisms involved. We uncover a central role for GATA transcription factors in
integrating and regulating Wnt signalling in cardiogenesis. GATA gene expression emerges
as the relevant target for inhibition by canonical Wnt/β-catenin signalling, since
experimentally reinstating GATA function overrides β-catenin-mediated inhibition and
restores cardiogenesis. Furthermore we find that GATA transcription factors in turn directly
regulate not only Nkx2-5 but also Wnt11. Using morpholino antisense oligonucleotides
designed against Wnt11, we show that Wnt11 function is indeed required to a large extent for
mediating the cardiogenesis-promoting activity of GATA transcription factors. These results
demonstrate that GATA transcription factors occupy a central position between canonical
and non-canonical Wnt signalling in a pathway regulating heart muscle differentiation.


The significance of the centrosomal localization of Xenopus Axin-related protein
for its function.
Evguenia M. Alexandrova and Sergei Y. Sokol.
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine,
New York, NY 10029, USA.

        The Wnt/β-catenin pathway regulates cell differentiation and proliferation in multiple
systems. However, the subcellular localization of Wnt pathway components and its
significance for the pathway regulation are not well understood. We found that Xenopus
Axin-related protein (XARP), a component of the β-catenin destruction complex, is localized
to the centrosome. This localization requires the carboxy-terminal region of XARP, including
the DIX domain. Mutations in several conserved amino acids result in a displacement of
XARP from the centrosome to the cytoplasm. However, this region alone is not sufficient for
centrosomal localization, as it is found in both the centrosome and cytoplasmic microtubules.
Other components of the β-catenin destruction complex, GSK3β, APC, and phospho-β-
catenin have been also reported to localize to the centrosome, suggesting that β-catenin
destruction may take place at the centrosome. Surprisingly, XARP mutants with poor
centrosomal localization revealed an enhanced ability to antagonize Wnt signaling. Thus, the
centrosome may inhibit XARP activity and promote Wnt/β-catenin signaling. In support of this
idea, Wnt3a, Xdsh and dFz1 displace XARP from the centrosome, possibly activating a
negative feedback loop. Our results contrast recently published reports showing a negative
regulation of the Wnt/β-catenin pathway by the basal body and primary cilium, originating
from the centrosome.

Role of Id proteins in Neurogenesis
Ana Alexandra Almeida1, Shin-ichi Ohnuma1,2 and Anna Philpott1
1
  Department of Oncology, University of Cambridge; 2Institute of Ophthalmology, University
College London

The cell cycle and cell fate determination are tightly regulated processes. However, the
molecular mechanism by which cell cycle regulators influence cell fate determination and
vice versa remain poorly understood. Id proteins, a class of helix-loop-helix (HLH)
transcriptional repressors, possess the dual function of acting as positive regulators of cell
proliferation and negative regulators of cell differentiation.
During development, the family of HLH transcription factors are key regulators of lineage
commitment and cellular differentiation. While the majority of basic helix-loop-helix (bHLH)
proteins drive the expression of lineage specific markers, Id proteins negatively regulate their
function by inhibiting DNA-binding through heterodimer formation.
We investigated the role of Ids in neurogenesis. Consistent with previous reports,
overexpression of Ids does not affect the expression of early neuronal markers such as
Sox3. However, we found that both Id2 and Id3 are able to disrupt the expression of the late
neural markers NeuroD and Neural-β-Tubulin. This ability of Id2 and Id3 to inhibit
differentiation of primary neurons can be rescued by co-overexpression with the bHLH
proneural protein Neurogenin (NGN). In addition, both Ids are able to inhibit neurogenin’s
ability to activate transcription in luciferase reporter assays. In vitro, Id3 significantly reduces
the DNA binding ability of NGN/E12 heterodimers. Interestingly, Id2 midly affects the binding
of NGN/E12 to DNA.
Taken together these results show that Id2 and Id3 negatively regulate NGN activity,
however through distinct mechanisms. Hence, clarifying the mechanisms by which Id2 and
Id3 block primary neurogenesis will contribute to the understanding of the biological role of
Ids in cell fate determination.


Functional analysis of EIG121L (estrogen-induced gene 121-like) in early Xenopus
development
Tetsuro Araki, Morioh Kusakabe, and Eisuke Nishida
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto
University, Kyoto 606-8502, Japan

 The bone morphogenetic protein (BMP) signaling pathway plays a pivotal role in epidermal
differentiation in the ventral ectoderm of early Xenopus embryos. Although it has been
reported that many secreted factors regulate the BMP pathway in the ectoderm, it is poorly
understood how the BMP pathway is activated in the ventral ectoderm. Here we report that a
novel transmembrane protein EIG121L (estrogen-induced gene 121-like) is a regulator of the
BMP pathway in the ventral ectoderm. EIG121L is an evolutionarily conserved gene, but its
function has not been examined in any organisms. We identified a Xenopus ortholog of
EIG121L (xEIG121L) as one of genes whose expression was downregulated by Ras
activation. Expression of xEIG121L was first detected in the ventral ectoderm at the gastrula
stage in Xenopus embryos. At the neurula stage, xEIG121L was expressed strongly in the
presumptive hatching and cement gland regions within the ectoderm surrounding the neural
plate. At the tailbud stage, xEIG121L expression was limited to the hatching gland. After
hatching, in tadpoles, xEIG121L was expressed in the head, heart and fin. Knockdown of
xEIG121L by using antisense morpholino oligonucleotides (MO) in early embryos caused
gastrulation defects, resulting in head and tail reductions. Expression of epidermal genes and
BMP/Smad1 signaling were markedly decreased in the ectodermal explants injected with
xEIG121L MO. These results show that xEIG121L is involved in BMP/Smad1 signaling in the
ventral ectoderm.

Jiraiya Inhibits BMP Signaling by Blocking Intracellular Transport of Type-II BMP Receptors
during Early Embryonic Patterning
Toshihiro Aramaki, Noriaki Sasai, and Yoshiki Sasai
Center for Developmental Biology, RIKEN, Kobe, Japan

Although a large number of previous studies have described extracellular and intracellular
controls of BMP signaling during early vertebrate embryogenesis, relatively little is
understood about the regulation at the BMP receptor (BMPR) level. Here we report the
endoplasmic reticulum (ER) membrane protein Xenopus Jiraiya, which plays an anti-BMP
role by limiting BMPR proteins on the cell surface. Maternal Jiraiya expression is widely
found on the animal side, and zygotic Jiraiya expression is exclusively observed in the neural
plate. MO-mediated knockdown of Jiraiya leads to phenotypes similar to those caused by
increased BMP signals: mesoderm ventralization, reduced neural differentiation and
increased dorsal-lateral markers in the neural plate. In the luciferase assay, Jiraiya inhibits
BMP4-induced luciferease activity in the animal cap while Jiraiya-MO enhances it. In
dissociated animal cap cells, Jiraiya reduces BMP4-induced phosphorylation of Smad1/5/8 in
a cell-autonomous fashion while Activin-induced phosphorylation of Smad2/3 is unaffected.
Jiraiya causes the retention of type-II BMPR proteins in the ER and selectively decreases
these receptor proteins on the surface. Thus, Jiraiya represents a novel regulatory
mechanism of the cellular responsiveness to BMP signals at the receptor level during early
embryogenesis.


A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus.
Aviad Keren, Anat Keren-Politansky and Eyal Bengal
Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences,
Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096,
Israel.

               Dorsal-ventral patterning of mesoderm is specified by signaling centers
secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen
gradient is translated intracellularly into fate decisions remains largely unknown. Here, we
report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm
patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient
along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the
phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm
dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-
VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high
levels of MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal
organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin
and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E
induces expression of lateral genes without inducing the expression of dorsal genes. Loss of
CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into
the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data
indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB
activities along the axis.


Paraxial Protocadherin function during gastrulation: cell adhesion, cell shape and
signaling

Corinna D. Berger and Herbert Steinbeisser
Institute of Human Genetics, University Heidelberg, INF 366, 69120 Heidelberg
corinna.berger@med.uni-heidelberg.de

The formation of tissues und tissue boundaries during gastrulation is a prerequisite for the
establishment of the body plan. In Xenopus the future mesendoderm involutes as a sheet of
cells, and the directed movement of individual cells leads to the coordinated movement of
this tissue. During this process the mesodermal cells move past the adjacent ectodermal
cells without mixing. Paraxial Protocadherin (PAPC) is first expressed at the onset of
gastrulation in the dorsal mesoderm and expands its expression domain to all of the
mesoderm except for the future notochord. Loss of PAPC leads to defects in both convergent
extension and tissue separation. On a cellular level we observe that after knock-down of
PAPC the mesodermal cells don’t acquire their bipolar cell shape. Cell polarity markers such
as PKCλ fail to become localized. Loss of PAPC also leads to reduced RhoA activity in the
dorsal mesoderm. Furthermore PAPC affects the distribution of Cadherin in both gain and
loss of function studies.

Gap junctional communication acts downstream of leftward flow in left-right axis
development
Tina Beyer and Martin Blum
University of Hohenheim, Stuttgart, Germany

Determination of the left-right (LR) body axis is essential for correct placement of the inner
organs, such that the heart points to the left, lung lobation occurs in a coordinated manner,
spleen and stomach develop on the left body side and the gut coils asymmetrically.
Disturbances of this arrangement, with the exception of complete mirror-image inversions,
invariably lead to severe diseases. Recently, we confirmed that a cilia-driven leftward flow
also acts in Xenopus laevis in the process of symmetry breakage, and that flow happens in a
monociliated structure called gastrocoel roof plate (GRP). Cilia rotate in a clockwise manner,
generating a leftward flow of extracellular fluid. If this Leftward Flow was blocked, LR-axis
determination was disturbed. As asymmetric gene expression initiates slightly after flow
stages in the left lateral plate mesoderm (LPM), and abrogation of flow prevents this
induction, one of the open questions concerns the mechanism by which an asymmetric
signal travels from the GRP to the LPM. An attractive candidate for signal transfer is cell-cell
communication via gap junctions (GJC), particularly because GJC is known to be an
important determinant of LR- development. To investigate the role of GJC in flow stage
embryos, inhibition experiments using heptanol were performed. These experiments
demonstrated that inhibition of GJC in late neurula-stage embryos, during and after flow-
stages, induced LR-defects and resulted in absent left-sided marker gene expression. In an
educated guess approach we looked for connexin genes potentially involved in this process.
We identified two gap junctional subunits expressed during neurulation. Knockdown
experiments confirmed involvement of these subunits, demonstrating that indeed GJC is
involved in induction of left-sided marker gene expression.


Rigorous probe-to-transcript mapping leads to significant improvement of Affymetrix
Xenopus laevis GeneChip probesets annotation
Biasci D., Giudetti G., Barsacchi G., Andreazzoli M.
Laboratory of Cell and Developmental Biology, Department of Biology, University of Pisa

Affymetrix GeneChip microarrays are a widely used platform for gene expression studies in a
broad variety of model systems. However, despite the evolution of biological databases and
the increasing number and availability of annotated sequences, at least 25% probesets of
Xenopus laevis and Xenopus tropicalis GeneChips still have little informative annotation
(referred to as Transcribed locus). Furthermore, previous studies have shown
inconsistencies on Affymetrix probeset-to-transcripts assignments in human and mouse
GeneChips (Dai et al. Nucleic Acids Res, 2005; Liu, Zeeberg et al. Bioinformatics 2007),
reasonably the most used in the scientific community. No exhaustive data about this issue
are available for Xenopus microarrays. Several online resources do exist to assess probeset
reannotation, but they rely on heavy complexity reduction and elaboration compromises.
These observations have made probeset reannotation an important issue for the correct
interpretation of microarray data.
To this purpose we developed BaseRank, a new fast indexing and alignment algorithm for
biological sequences that we used to perform rigorous alignments of all the Affymetrix
Xenopus laevis chip probeset sequences against all available transcript sequences in the
latest databases’ releases.
With a considerable reduction of elaboration time, compared to similar algorithms, we thus
generated a complete transcript-level annotation table for Xenopus laevis, that helped us to
better assess and refine our microarray results.
A wider task using the aforementioned algorithm is currently underway to obtain a up-to-date
annotation table for Xenopus laevis 2.0 and Xenopus tropicalis Affymetrix expression arrays
based on transcriptome and genomic information.




Cell fate determination in neural retina by NM23 members
Aikaterini Bilitou1, Toshiaki Mochizuki1 and Shin-ichi Ohnuma1, 2,
1
  Hutchison/ MRC Research Centre, Department of Oncology, University of Cambridge, Hills
Road, Cambridge CB2 0XZ, UK
2
  Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V
9EL, UK

The NM23 (Non-metastatic) family is almost universally conserved across all three domains
of life. The first members were identified in a tumour suppressor gene screen of metastatic
human cell lines and a screen for abnormal imaginal wing disc development in Drosophila,
suggesting their role in tissue homeostasis and development. Although nucleoside
diphosphate kinase activity (NDPK) has been attributed to many NM23 members as primary
function, studies show that they also possess other functions in the cell, for instance acting
as protein kinases or transcription factors. In this study, we report the role of NM23 members
in neural development and in particular retinal fate determination. We describe the
expression pattern and functional analysis of NM23 in the African clawed frog, Xenopus
laevis, with interest in the neural retina. In vertebrates, neural cell fates are influenced before
or around the final cell cycle. Interestingly, NM23 members are expressed in the neural retina
in distinct and overlapping patterns. We have identified NM23-X4 as a binding partner of
Xenopus cyclin dependent kinase inhibitor, p27Xic1. In Xenopus retinogenesis, p27Xic1
functions as a determinant of Müller glial cells in addition to its role as a cell cycle inhibitor. In
neural retina, the expression of NM23-X4 and NM23-X3 overlaps spatially with p27Xic1
expression in the ciliary marginal zone at a timing coincident with the events of cell fate
determination. Our in vivo functional analysis has revealed that NM23-X4 activates
gliogenesis in the retina and co-overexpression of NM23-X4 with p27Xic1 inhibits p27Xic1-
mediated gliogenesis. These findings suggest that NM23X4 functions in the retina as an
inhibitor of gliogenesis through interaction with p27Xic1.
Identification of Downstream Targets of Ptf1a. C. Bilogan, Z. Jarikji, M. Horb. Division of
Experimental Medicine, McGill University; Institut de recherches cliniques de Montréal
(IRCM), Montréal, Québec H2W 1R7 Canada

         Ptf1a is a basic helix-loop-helix (bHLH) transcription factor complex that is essential
for development of both endocrine and exocrine pancreas. Our lab recently published a
detailed analysis of Ptf1a function in Xenopus development. In order to identify downstream
targets of Ptf1a we performed a microarray analysis between control pancreatic tissue and
pancreatic tissue lacking Ptf1a. This analysis revealed that a partial knockdown of Ptf1a
function resulted in a complete loss of acinar cells, but had no effect on endocrine cells, while
a complete knockdown affected both endocrine and exocrine cells. How Ptf1a functions to
specify endocrine as well as exocrine cells is currently not known. One particular gene of
interest, which was identified in the microarray, is Staufen2, an RNA binding protein first
identified in Drosophila as an essential factor required for germ line specification and neural
development. Staufen2 expression was normal in the partial knockdown of Ptf1a but was
entirely absent in the complete knockdown of Ptf1a, suggesting a putative role in endocrine
cell development. We examined the expression of Staufen2 and determined it to be localized
in the dorsal pancreas, where endocrine cells originate, by in situ hybridization. The Staufen2
promoter also contains many putative pancreatic transcription factor sites including Ptf1a and
NeuroD. Along with these results, loss-of-functions studies on Staufen2, using morpholino
technology, result in abnormal expression of many pancreatic cell types within the affected
pancreas. These preliminary results further support our hypothesis that Staufen2 may be
involved in endocrine cell fate specification. In this poster we will present our initial
investigation into downstream targets of Ptf1a and their role in pancreas cell fate
determination.



A role for XtSulf1 in germ cell migration
Nicholas D. Bland, Wendy M. Moore & M.E. Pownall
Department of Biology (area 11), University of York, York, UK, Yo10 5YW

        Sulf1 is a 6-o-endosulfatase which specifically removes sulphate moieties from the
sugar side chains of heparan sulphate proteoglycans (HSPG). HSPGs bind avidly to many
different proteins and can facilitate the interaction between ligands and their receptors at the
cell surface. This includes members of the fibroblast growth factor (FGF), bone morphogenic
protein (BMP) and WNT signaling pathways. By modifying the sulphation pattern of HSPGs,
Sulf1 can modulate the ability of ligands to interact with their receptors. Zygotic XtSulf1 is
expressed widely and dynamically during Xenopus tropicalis development. Maternal
XtSulf1transcripts are deposited in the egg and co-.localise with known markers of the germ
plasm.
        Cells allocated to the germ line during cleavage stages are characterized by the
inheritance of germ plasm. Specific mRNAs are recruited to the germ plasm by sequences in
their 3’UTR. Further development of the germ line involves a significant migration of the
presumptive germ cells. The presumptive germ cells (PGCs) reside in the endoderm until
tailbud stages and then migrate through the endoderm first laterally, then dorsally and
anteriorly. Ultimately, the PGCs migrate through the dorsal mesentery and into the genital
ridges.
        Here we described the expression of XtSulf1 in the germ line. We also examine any
role for XtSulf1 in the migration of the presumptive germ cells by looking at the effect of
XtSulf1 overexpression on PGC migration using the germ plasm markers XtCat2, XtDaz and
XtPat1.
The homeobox gene Goosecoid acts as a regulator of Planar Cell Polarity (PCP)
Bärbel Ulmer, Philipp Andre, Kirsten Deißler, Verena Mauch, Axel Schweickert and Martin
Blum
University of Hohenheim, Institute of Zoology, Stuttgart, Germany

Goosecoid (Gsc) was the first organizer-specific gene cloned from Xenopus. Ventral
injections induced secondary axis formation with high frequency. The Gsc knockout mouse,
however, surprisingly revealed no gastrulation defects. To study Gsc in the mouse by gain-
of-function, Gsc was conditionally misexpressed under the control of the Brachyury streak
enhancer. Mid-gestation embryos displayed neural tube closure defects (NTD) with about 20
% penetrance upon mild misexpression. Massive misexpression resulted in embryonic
lethality at mid-gastrula stages. Notochord formation and dorsal extension were
compromized in affected embryos. Specificity was demonstrated by massive NTD in
chimaeric mouse embryos generated with ES cells which massiveley overexpressed Gsc. In
order to investigate whether this result was a mouse-specific effect, we misexpressed Gsc
protein from an inducible Gsc-GR construct in Xenopus. Dorsal injections induced NTDs at
very high frequency when Gsc was activated between stage 6 and 11, i.e. before and during
gastrulation. Marker gene analyses showed, that NTDs were not due to patterning defects.
As NTDs were reminiscent of phenotypes induced upon interference with the PCP-Pathway,
we hypothesized that Gsc might act as a transcriptional regulator of PCP. Therefore,
downstream components of PCP were assayed for their potential to rescue Gsc-induced
NTD. Indeed, the three components analyzed so far, constitutively active RhoA, strabism and
prickle all were able to partially revert Gsc phenotypes with high statistical significance.
These unexpected findings might help to explain the lack of a gastrulation phenotype in
knockout mice. In addition, we like to suggest a role for the endogenous Gsc gene in
prechordal mesodermal cells, i.e. in switching from convergent extension (notochord) to
migration (prechordal mesoderm).

Protein Tyrosine Kinase 7 (PTK7) interacts with Wnt Signaling to Regulate XMeis3
expression in the Developing Xenopus Nervous System
Naama Blum and Dale Frank, Department of Biochemistry, Rappaport Institute for Research
in the Medical Sciences, Technion Medical School, Haifa, Israel.

Protein tyrosine kinase 7 (PTK7) is a recently identified novel regulator of vertebrate planar
cell polarity (PCP) affecting neural plate convergent-extension (CE) and neural tube closure
in mouse and Xenopus embryos. PTK7 is expressed in the Xenopus gastrula-neurula stage
neural plate region. Our previous studies showed that the caudalizing XMeis3 transcription
factor plays a role in regulating neural CE. XMeis3 induced convergent extension was
dependent on the presence of FGF/MAPK and Wnt-PCP signaling pathways. In initial studies
we examined XMeis3/PTK7 epistasis to determine if PTK7 expression is downstream to
XMeis3 in the process regulating neural CE. In XMeis3 morphant embryos that have
impaired neural CE, PTK7 mRNA is expressed at normal levels. In contrast, in PTK7
morphant embryos, there is a sharp reduction of XMeis3 expression. Similar to XMeis3
morphant embryos, PTK7 morphant embryos also have a reduction in posterior neural
markers expressed in hindbrain, primary neuron, and neural crest cell types. Studies in our
lab have shown that XMeis3 expression is highly dependent on canonical Wnt signaling. We
thus examined if PTK7 knock down could also alter canonical Wnt signaling (in addition to
Wnt-PCP). Animal cap explants co-expressed Wnt3a mRNA and the PTK7 MO. In these co-
injected explants, we observed a strong reduction in Xnr3 and siamois expression. These
results suggest that in addition to its role in Wnt-PCP signaling, PTK7 plays a role in
regulating canonical Wnt signaling.
Roles for GSK-3 in the anterior patterning of the neural crest.


Triona G. Bolger and Karen J. Liu
Department of Craniofacial Development, Kings College London

Cephalic neural crest gives rise to many different cell types including bone, connective tissue
and pigment cells. Glycogen synthase kinase 3 (GSK-3) is known to play roles in many
developmentally important signaling pathways such as Wnt. GSK-3β mouse mutants survive
gestation and die soon after birth with craniofacial defects, suggesting the importance of
GSK-3 in neural crest development. The late lethality of the GSK-3 mutant phenotype is
surprising due to the involvement of GSK-3 in so many pathways. This suggests that GSK-
3α, a closely related serine threonine kinase, may compensate for the loss of GSK-3β in
embryonic development. We are taking advantage of the ease of manipulation of early
Xenopus embryos to address this question and to ask whether GSK-3α has unique
functions, focusing on the role of GSK-3 in setting up the neural crest pattern and the
migration of neural crest cells. We have identified Xenopus laevis GSK-3α and are
comparing its sequence, expression and activity to that of Xenopus laevis GSK-3β. Using
GSK-3 inhibitors, we are testing our hypothesis that GSK-3 is important for the migration of
neural crest by observing effects on the development of anterior structures such as the
neural crest, the developing brain and the preplacodal region.




Role of miRNA-9 in neurogenesis
Boyan Bonev, Matthew Ronshaugen and Nancy Papalopulu
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

        MicroRNAs (miRNAs) belong to a class of small non-coding RNAs (21-25nt), which
are involved in the regulation of gene expression at the post-transcriptional level. They have
been shown to be important in animal development, particularly in the patterning of the
central nervous system (CNS). One of the most evolutionary conserved miRNA in the CNS
is miR-9, but its precise role is unclear. Here, we show that the expression of the primary
miR-9 genes differ both spatially and temporally in developing Xenopus embryo: miR 9a-1 is
localized primarily to the eye, forebrain, mid- and hindbrain and is highly enriched to the
ventricular zone, while the expression of miR 9a-2 and miR 9-3 is weaker and appears to be
present around the neural tube and in a patch of cranial tissue. Overexpression of miR-9
causes lateral expansion of the brain and increased neuronal differentiation, predominantly in
the dorsal midbrain. Blocking endogenous miR-9 with a morpholino leads to decreased
neuronal differentiation, but no changes in brain morphology. One of the potential miR-9
targets in the forebrain is FoxG1, which is a transcription factor involved in the development
of cortical progenitors. Other predicted targets of miR-9 include the basic helix-loop-helix
transcription factor Hes-1 and the regulator of alternative splicing nPTB. We are also
currently investigating the potential role of miR-9 as a link between TGF-β and Wnt
signalling. Our findings indicate that miR-9 is involved in Xenopus neurogenesis, possibly
through regulation of the cell cycle and/or commitment of neuronal progenitors.


High-Resolution Studies of Xnr1 Signaling and Left-Right Asymmetry in Xenopus
Lindsay T. Bramson1,2 and Christopher Wright1,2
1
  Vanderbilt University Medical School, 2Program in Developmental Biology
3144 MRBIII 465 21st Avenue S, Nashville, TN 37232
Vertebrate left-right (L-R) asymmetric morphogenesis is established by a Nodal-initiated left-
sided signaling cascade within the lateral plate mesoderm (LPM). Much evidence supports
the hypothesis that a Nodal autocatalytic loop and a Nodal-induced negative feedback loop
work together to coordinate the spatiotemporally dynamic morphogenesis between both
sides of the embryo. The largest gaps exist in understanding the cellular and tissue
patterning events that precede asymmetric organ placement, as well as the routes of transfer
of Nodal (inducer) and Lefty (inhibitor) ligands. A substantial hurdle is the inability to detect
the endogenous proteins. I have initiated a two-pronged approach to characterize LPM
architecture and the transport characteristics of Nodal and Lefty within and away from the
LPM. High-resolution analysis of the LPM before, during and after asymmetric signaling
indicates that the L and R LPM are bilayered, express some initial epithelial markers, and are
sandwiched by a rich extra-cellular matrix (ECM). Functional epitope-tagged proteins and
tissue grafting experiments are revealing that a Myc-epitope-tagged Xlefty can be detected at
and perhaps beyond these ECM-rich interfaces. I will present ideas on how these findings
will be extended, to test how the ECM acts as a barrier or facilitator of long-range signal
transfer.




Analysis of the role of the B56α regulatory subunit of protein phosphatase 2A in β-
catenin degradation.
Aron D. Branscomb, Eve Santos, and Joni M. Seeling
Department of Biology, City University of New York, Queens College, Flushing, USA.

    A Xenopus laevis egg extract system is an ideal system to analyze Wnt pathway activity,
as it recapitulates essential features of Wnt signaling initially characterized in other systems.
Protein phosphatase 2A (PP2A) consists of a catalytic C, a structural A, and a regulatory B
subunit, and we found that the B56α regulatory subunit of PP2A inhibits Wnt-dependent
reporter activity and reduces Wnt-induced secondary body axes in Xenopus laevis. In
addition, PP2A C and B56α subunits promote the degradation of β-catenin in Xenopus egg
extracts. We found that a carboxy-terminal region of B56α, B56α291-486, binds to
adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, but not to PP2A A,
while an amino-terminal region of B56α, B56α1-343, binds to PP2A A but not to APC.
However, the region of B56α involved in promoting β-catenin degradation has not been
investigated. To better understand the functional domains of B56α, we have examined how
various B56α mutants affect Wnt signaling in a β-catenin degradation assay using Xenopus
egg extracts.

Characterization of Peter Pan during neural development of Xenopus laevis
Verena Bugner, Susanne Gessert and Michael Kühl
Institute for Biochemistry and Molecular Biology, Albert-Einstein-Allee 11, D-89081 Ulm
Email: verena.bugner@uni-ulm.de
We have recently shown that Pescadillo is a potential target gene of Xenopus non-canonical
Wnt-4 signaling (Gessert et al., 2007, Maurus et al., 2005). We here provide a first functional
characterization of Peter Pan (PPan), a Pescadillo interacting protein. PPan was first
identified in Drosophila melanogaster. In this organismn, PPan mutant larvae show growth
defects and minimal DNA replication (Migeon et al., 1999). PPan belongs to a highly
conserved gene family, including homologues in different species, containing a BRIX
domain. Here, we want to investigate for the first time the function of PPan during Xenopus
laevis neural development. Analysis of the spatial expression pattern shows that PPan is
expressed in anterior neural tissue and later in the developing eye, the branchial arches, the
brain and in the somites. Loss of PPan function by use of a specific antisense morpholino
oligonucleotide leads to a severe eye and cartilage phenotype. Marker gene analysis
revealed a reduction of early eye marker genes Rx, Pax6 and Otx2 as well as neural crest
marker genes such as Twist, Slug and FoxD3 upon loss of PPan function. In contrast, the
expression of the pan neural marker gene Sox3 and the brain marker genes Emx1, En2 and
Krox20 seems to be unaffected. Future experiments will show if PPan is also involved in cell
cycle regulation during Xenopus development.



Molecular mechanism of cardiac patterning and morphogenesis
Simona Caporilli and Branko Latinkic
Cardiff University, Genetic Department, Museum Avenue, CF10 3US, Cardiff, UK.

Vertebrate heart development involves a precise sequence of morphogenic events from
which a complex structure is formed from a linear heart tube.
To study the heart development in mammals is difficult because most alteration of heart
structure will be lethal. Therefore we use an alternative model Xenopus laevis. Ectopic gene
expression can be achieved in frog embryos by simple microinjection of sense RNA.
Embryos can survive for a greater development period than mammals without a fully
functional heart.
It has previously been established that it is possible to induce differentiation of cardiac tissue
in Xenopus animal caps explants previously injected with sense mRNA.

The aim of this project is to understand the mechanism that regulates the anterior-posterior
pattern establishment. This determines the formation of the ventricular and atria chambers
and the mechanisms which regulate heart morphogenesis.

It has previously been established that it is possible to induce differentiation of cardiac tissue
in animal cap explants which were previous injected with mRNA. Using a “cardiogenic assay”
we show that is also possible to obtain expression of ventricular specific marker in the
explants which normally are expressed at late stage of development.
We are interested to discover if cardiomyocites non-positive to ventricular marker expression
are present in these explants. This can help us to understand if atrial specification is lead by
other myocites cell-types.
Using a “heart formation assay” we show that is possible to obtain a secondary beating heart
in a host embryo from a group of animal cap explants previously injected with mRNA. We
used this assay to determine spatial and temporal variability of the host embryo to permit the
formation of a secondary beating heart.



Role of sonic hedgehog signalling in the development of the branchial arches

Alejandra Paganelli, Grazielle Pacheco Silva, Silvia López and Andrés Carrasco

Laboratorio de Embriología Molecular. Instituto de Biología Celular y Neurociencias.
Facultad de Medicina, UBA. Paraguay 2155 Piso 3 (1121) Ciudad Autónoma de Buenos
Aires. Argentina

The neural crest constitutes a multipotent cell population, which gives rise to a broad range
of derivatives, including pigment cells, craniofacial skeleton, connective tissues, neurons and
glia of the peripheral nervous system. In Xenopus, three major populations of migrating
cranial neural crest cells originate at distinct axial levels of the mid- and hindbrain and target
the mandibular (first), hyoid (second) and the third and fourth branchial arches.
Sonic hedgehog overexpression leads to an enlargement of the visceral and branchial
arches and to an up-regulation of Fgf-8 in ectodermal cells. Conversely, knocking down Shh
signalling with a morpholino antisense oligonucleotide leads to a complete disorganization of
the arches and to a down-regulation of Fgf-8. These results indicate that Shh is necessary
for the correct formation and patterning of the visceral and branchial arches.



Dkk-1 regulates a complex containing both Wnt5a and Wnt11, which activates
canonical and non-canonical signaling in Xenopus axis formation
Sang-Wook Cha, Emmanuel Tadjuidje, Qinghua Tao, Christopher Wylie and Janet Heasman
Division of Developmental Biology, Cincinnati Children’s Research Foundation, 3333 Burnet
Avenue, Cincinnati, Ohio 45229-3039

Wnt signaling in development and adult tissue homeostasis requires tight regulation to
prevent patterning abnormalities and tumor formation. Here we show that maternal Dkk-1
both prevents canonical Wnt signaling in ectopic sites in the embryo and regulates excessive
canonical Wnt signaling in the correct location. More surprisingly, Dkk-1 also antagonizes
non-canonical Wnt signaling, as evidenced by Dkk-1/b-catenin loss of function experiments
where Dkk-1 depletion caused increased JNK-1 phosphorylation and enhanced
morphogenetic movements regardless of the absence of b-catenin. Since maternal Wnt5a
loss of function phenocopied Wnt11 loss of function, and loss of either ligand prevented the
upregulation of both canonical and non-canonical signaling caused by Dkk-1 depletion, we
conclude that maternal Wnt5a, as well as Wnt11, is required to activate canonical and non-
canonical signaling.
We show that both Wnt11 and 5a are required for signal transduction in loss of function
studies, and they synergize when over-expressed. Co-immunoprecipitation experiments
show that the fraction of the Wnt11-HA and Wnt5a-myc protein pool that interacts, consists
of dimers and oligomers but not monomers, which taken together with the functional data,
suggests that complexes, not monomers, are active in signaling.
This work reveals a new aspect of Wnt signaling, that of two Wnts acting in a complex
together to regulate embryonic patterning.



Regulation of early Xenopus development by polycomb group proteins

Megan Cox, Susan M. Johnson, Jason Fletcher, Chenbei Chang
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294

Polycomb group (PcG) proteins were first identified in Drosophila as crucial factors for stable
and heritable repression of homeotic genes. They form two Polycomb repressive
complexes, PRC1 and PRC2, which modify histones and chromatin structures for epigenetic
repression of the target Hox genes. Vertebrate PcG proteins are then identified and shown
to modulate stem cell pluripotency, genomic imprinting, X-inactivation and Hox gene
expression. However, their roles in early vertebrate embryonic induction and patterning are
not completely understood. In this study, we used gain- and loss-of-function approaches to
examine the activities of EED and EZH2, both of which are key components of PRC2, in
early embryogenesis of Xenopus embryos. We show that EED and EZH2 are expressed
widely in frog gastrulae, and its transcripts are maintained in the neural tissues at the tailbud
and tadpole stages. Alteration of EED or EZH2 protein level in ectodermal cells via
overexpression or antisense morpholino oligonucleotide depletion affects neural crest
development as well as anterior-posterior neural patterning; while perturbation of EED or
EZH2 level in dorsal marginal zone cells leads to gastrulation defects. Our data indicate that
EED and EZH2 play a critical role in patterning and morphogenesis of early frog embryos by
regulation of the expression of both Hox and non-Hox genes.


C/EBPalpha initiates myelopoiesis in pluripotent embryonic cells

Yaoyao Chen, Ricardo Costa, Martin Roth and Enrique Amaya

The Healing Foundation Centre, Faculty of Life Science, University of Manchester,
Manchester M13 9PT, UK

The molecular mechanisms that underlie the development of primitive myeloid cells in
vertebrate embryos are not well understood. Here we characterize the role of cepba during
primitive myeloid cell development in Xenopus tropicalis. We show that cepba is the first
known hematopoietic factor expressed in the embryo. Loss and gain-of-function studies
show that it is both necessary and sufficient for the development of functional myeloid cells.
In addition, we show that cepba misexpression leads to the precocious induction of myeloid
cell markers in pluripotent prospective ectodermal cells and this occurs without the cells
transitioning through a mesodermal state. Finally we use live imaging to show that cepba
expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as
highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds
and the ability to enter the circulation. Thus cepba is the first known single factor capable of
initiating the entire myelopoeisis pathway in pluripotent cells in the embryo.

Identification of a novel negative regulator for activin-like signaling
Seong-Moon Cheong, Hyunjoon Kim and Jin-Kwan Han
Department of Life Sciences, Pohang University of Science and Technology, San 31, Hyoja
Dong, Pohang, Kyungbuk, 790-784, Republic of Korea

Abstract
The signaling molecules that regulate the specification of three germ layers in early
embryogenesis remain to be determined further. Here we show that Xdpcp (Xenopus dok-
PTB containing protein) has a pivotal role for the regulation of mesoderm formation in
Xenopus and negatively regulates the activin-like signaling pathway. We isolated cDNA for
Xdpcp by using PCR-based method and sequence information in EST database and
examined its potential role in Xenopus embryogenesis. It is strongly expressed in the animal
hemisphere at the cleavage and blastula stages. Overexpression of Xdpcp RNA causes
defects in mesoderm formation. In addition, loss of Xdpcp function by injection of morpholino
oligonucleotides (MO) leads to expansion of the mesodermal territory. Moreover, we found
that the secondary axis formation by ventrally forced expression of activin is recovered by
coexpression of Xdpcp and it inhibits the phosphorylation and nuclear translocation of
Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor
and inhibits activin-like signaling by disturbing the interaction between Smad2 and Alk4.
Taken together, these results indicate that Xdpcp is essential for mesoderm specification by
regulating activin-like signaling pathway.

Ginsenoside Rg3 inhibits L-type Ca2+ channel currents by interacting with the Leu427,
Asp428, and Leu431 of transmembrane domain IS6

Sun-Hye Choi, Tae-Joon Shin, Byung-Hwan Lee, Mi Kyung Pyo, Sung-Hee Hwang,

Jun-Ho Lee, Sang-Mok Lee and Seung-Yeol Nah

Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-
701 Korea
We previously demonstrated that ginsenoside Rg3 (Rg3) inhibits L-type Ca2+ channel
currents with stereospecific manner and that Rg3-mediated inhibitions of L-type Ca2+ channel
currents are coupled to neuroprotection in rat cortical neurons, pointing to the presence of
specific interaction site(s) for Rg3 to regulate L-type Ca2+ channel in neuroprotection.
However, it is yet unknown how Rg3 interacts with L-type Ca2+ channel proteins. In the
current study, we sought to identify this site(s) in Xenopus oocytes expressing Ca2+ channel
mutants using the two-microelectrode voltage-clamp technique and site-directed
mutagenesis. To this end, we first assessed how point mutations of various amino acid
residues of the α1C-type Ca2+ channel affected Rg3 action. The mutations L42R, N428R or
L431K in transmembrane domain-1-segment 6 (IS6) of channels significantly attenuated the
Rg3 effect and caused a rightward shift of the Rg3 concentration-response curve compared
to wild-type Ca2+ channel currents. Further, double mutations L42R, N428R or L431K
abolished Rg3 action. Diltiazem, a L-type Ca2+ channel blocker, did not occlude Rg3 action in
wild-type and mutant channels, indicating that Rg3 does not share a common binding site or
pathway with diltiazem in wild-type and mutant channels. Mutations of many other residues,
including dihydropyridine binding sites, were without any significant effects. These results
indicate that Leu427, N428 and L431 in transmembrane IS6 play important roles in modifying
Rg3-mediated the α1C-type Ca2+ channel properties.



Vertebrate CASTOR is Required for Differentiation of Cardiac Precursor Cells at the
Ventral Midline
Kathleen S. Christine 1,2 and Frank L. Conlon 1,2,3
1
  Carolina Cardiovascular Biology Center, 2 Department of Biology, and 3 Department of
Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC

The CASTOR (CST) transcription factor was initially identified for its role in maintaining stem
cell competence in the Drosophila dorsal midline. We have shown that Xenopus CST is
expressed in the developing heart and is required for cardiogenesis. In CST-depleted
embryos, cardiomyocytes at the ventral midline fail to differentiate and are maintained as
cardiac progenitors, while cells in more dorsal regions of the heart undergo their normal
program of differentiation. Our fate mapping studies reveal that this ventral midline
population of cardiomyocytes ultimately gives rise to the outer curvature of the heart;
however, CST-depleted midline cells over-proliferate and remain a coherent population of
nonintegrated cells positioned on the outer wall of the ventricle. These midline-specific
requirements for CST suggest the regulation of cardiomyocyte differentiation is regionalized
along a dorsal-ventral axis and that this patterning occurs prior to heart tube formation.


A NARROW TEMPORAL INTERVAL FOR JAW SPECIFICATION DURING XENOPUS
DEVELOPMENT
Maxence Vieux-Rochas, Kamal Bouhali, Sabrina Baudry, Laurent Coen and Giovanni Levi
Evolution des Régulations Endocriniennes, CNRS, UMR5166, Muséum National d'Histoire
Naturelle, 7 rue Cuvier, 75231 Paris Cedex 05, France.

Abstract :
Cartilagineous and dermatochranial elements of the jaws are formed by Hox-negative
Cephalic Neural Crest Cells (CNCCs) emigrating from the prosencephalic and anterior
mesencephalic neural folds, and colonizing the first Pharyngeal Arch (PA1). Before migration
CNCCs lack the topographic information needed to unfold the jaw morphogenetic program.
They receive these instructions from endoderm and the ectoderm of PA1. This signaling
process results in the specification of the maxillo-mandibular territories of PA1. Endothelin-1
(Et1) signaling directs CNCC towards a mandibular identity while Fgf8 specifies a maxillary
identity. Dlx genes are a family of homeobox transcription factors which play a central role in
directing the morphogenetic program of CNCC. Et1 activates Dlx5 and Dlx6 in the
mandibular arch while Fgf8 activates Dlx1 and Dlx2 in the maxillary arch. Targeted
inactivation of Dlx genes in the mouse has shown that their combinatorial expression is at the
origin of jaw specification.
We treated with RA early developmental stages of Xenopus Laevis ranging from NF15 to
NF23 and we analysed the molecular, morphological and skeletal consequences of these
treatment on later stages. Craniofacial malformations were obtained but no effects on axis
formation were detected. This finding suggests that RA-sensitive molecular regulations
involved in jaw specification are taking place in this narrow developmental window. Both in
mice and tadpoles RA perturbs selectively craniofacial development when administered in a
temporal interval corresponding to the end of CNCC migration. This suggests a similarity in
the cellular and molecular strategy of jaw specification adopted by two species with a very
different mode of craniofacial development. Actually while in mice PAs are external
appendages, they are internal in Xenopus tadpoles and the topography of CNCC migration
and signalling is profoundly different between the two species. Patterning modification of a
single molecule can cause severe modification in skull development. This can be seen as an
example of how modulation of a single regulatory pathway can lead to the appearance of
multiple morphotypes, suggesting an explanation for phenotypic radiation and rapid
evolutionary changes.



The role of INSIGs and SREBPs in cholesterol biosynthesis,
Anne Corsing, Dörte Adolph, Thomas Hollemann, University of Halle/Wittenberg, Inst.
Physiol. Chemistry, Germany

Intracellular cholesterol is regulated by an elaborated negative feedback mechanism, which
senses the cholesterol level, and in consequence modifies the transcription and thus the
expression of proteins playing a fundamental role in synthesis, import and transport of
cholesterol. Two main actors of the machinery controlling cholesterol homeostasis are
sterol-regulatory-element-binding-proteins (SREBPs) and insulin-induced genes (INSIGs).
Human SREBPs form tight complexes with SCAP, a membrane bound protein of the ER. If
the cholesterol level decreases, this complex is incorporated into vesicles and travel to the
Golgi, where SREBPs are processed. In the nucleus, SREBP force the transcription of the
HMG-CoA-Reductase (Nohturfft et al.2000) and other important genes of cholesterol
homeostasis are activated. When cholesterol is found in excess, the SCAP/SREBP complex
is sequestered by INSIG-type proteins located at the membrane of the ER, resulting in a
reduced rate of cholesterol and fatty acid synthesis (Yang et al.,2002).
Here we report on our latest results on the role of Xenopus SREBP 2, INSIG 1 and INSIG 2
during early embryonic development of Xenopus laevis. We performed first loss- and
gain-of-function experiments to understand their role for the generation of the three germ
layers. Temporal and spatial pattern were analysed by whole-mount in situ hybridisation and
RT-PCR. We demonstrate that the expression of INSIGs and SREBPs are maternal,
declines until neurogenesis and increases again in further development. PCR showed that
SREBPs and INSIGs are ubiquitously expressed in adult tissues. Cell culture analyses will
complete our investigations of the function of SREBPs and INSIGs in the cellular regulation
of cholesterol homeostasis and their function in general embryonic development in Xenopus
laevis.




spib is required for primitive myeloid development in Xenopus
Ricardo Costa, Ximena Soto, Yaoyao Chen, Enrique Amaya

The Healing Foundation Centre, Faculty of Life Science, University of Manchester,
Manchester M13 9PT, UK

        We are very interested in the study of primitive hematopoiesis and the role that the
first embryonic blood cells have during development. Vertebrate blood formation is a
continuous process that starts soon after the end of gastrulation. Hematopoietic
development and cell differentiation then reaches steady-state homeostasis in the
mammalian bone marrow that is well characterized. The development of the first blood cells
and in particular primitive myeloid blood cells has received far less attention. In Xenopus
primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the
mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse
video microscopy, we recorded the migratory behaviour of primitive myeloid cells from their
birth. We show that these cells are the first blood cells to differentiate in the embryo and that
they are efficiently recruited to embryonic wounds, well before the establishment of a
functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically
expressed in primitive myeloid precursors. Using spib antisense morpholino knock down
experiments, we show that spib is required for myeloid specification, and, in its absence,
primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we
conclude that spib sits at the top of the known genetic hierarchy that leads to the
specification of primitive myeloid cells in amphibians.


Functional characterization of the Xenopus Wnt antagonist XsFRP5 in organ
development
Katharina Damianitsch and Tomas Pieler
Department of Developmental Biochemistry, GZMB, University of Göttingen

The primitive gut tube gives rise to a diverse set of internal organs along the anterior-
posterior axis. Studies in mouse, fish and chicken have revealed that many different signal
transduction pathways are involved in the patterning of the embryonic gut tube. We use
Xenopus laevis as model system to analyze the function of Wnt signaling during
organogenesis. Secreted Wnt antagonists are supposed to modulate Wnt activity leading to
more refined gradients of morphogen signaling. The secreted Wnt antagonist XsFRP5, which
is expressed in the liver diverticulum and subsequently at the gastric-duodenal border
(Pilcher and Krieg, 2002), was assayed for its function on endodermal organ specification
and delineation. Xenopus Wnt proteins interacting with XsFRP5 were identified by co-
immunoprecipitation experiments and further analyzed by scoring the inhibition of Wnt-
mediated axis duplication by XsFRP5 in Xenopus embryos, where XsFRP5 was able to
inhibit both canonical and non-canonical Wnts. A knock-down of XsFRP5 using antisense
Morpholino oligonucleotides resulted in a size reduction of the ventral pancreatic bud, as
assayed by the expression of the pancreatic precursor markers Ptf1a/p48 and XlHbox8. Liver
development proceeds unaffected upon loss of XsFRP5. Overexpression studies of XsFRP5
were performed in ectodermal pluripotent precursor cells, which were directed to an
endomesodermal fate by injection of VegT. Co-expression of increasing doses of XsFRP5
led to an increase in gene expression of pancreatic precursor as well as differentiation
markers. We propose that XsFRP5 plays a role in the delineation of developing organs at the
liver-ventral pancreas and stomach-duodenal borders.


PDGFA is required for orientation and directional migration of the mesendodermal
cells in the Xenopus gastrula,
 Erich W. Damm and Rudolf Winklbauer, Department of Cell & Systems Biology, University
of Toronto, Toronto, Canada, M5S 3G5
The cells of the involuting dorsal mesendoderm are thought to undergo intercellular migration
throughout gastrulation, however the pattern of migration of these cells has not been
established. Mesoderm migration is an important process during gastrulation and its
disruption results in abnormal head structures, shortened body axis and spina bifida. Using
scanning electron microscopy, we show that the mesendodermal cells are predominantly
unipolar and orient dorsally in the embryo. Timelapse recordings of combined mesendoderm-
blastocoel roof explants show that cells actively migrate, with their direction being determined
by the blastocoel roof. This pattern of movement is observed in both anterior and posterior
mesoderm. Furthermore, we show that disruption of platelet-derived growth factor A (PDGF-
A) function by microinjection of morpholino oligonucleotides or dominant negative RNA
constructs prevents the cells from adopting their dorsal orientation and inhibits directional
migration in explants. This effect of PDGF-A loss of function can be reversed by co-injection
of mRNA encoding the wild-type short form of PDGF-A. Altogether, our data suggest an
instructive role for PDGF-A as a chemoattractant in the directional migration of
mesendodermal cells. PDGF ligands and receptors are known for their roles in mitogenesis,
angiogenesis, cell death regulation, and directional cell migration. These molecules are also
known to be upregulated in certain cancers and may play a role in invasiveness. This
research contributes to a further understanding of the process of intercellular migration of
dorsal mesendoderm during gastrulation in Xenopus.

Transcriptional control of eye and brain development: Otx2 and Sox2 coregulation
Hiroki Danno, Tatsuo Michiue, Shoichi Ishiura, Makoto Asashima
University of Tokyo

Central nervous system of vertebrates develops from the neural plate of the neurula embryo.
The subdivision of neural plate into individual parts of brain and eye is controlled by many
transcription factors and transcriptional cascades. However, the direct linkages among
individual transcription factors remain unclear. Recently, we found the molecular links among
three transcription factors associated with eye development. Initially, we explored the
upstream regulation of retina and anterior fold homeobox gene Rax (also known as Rx1) by
the comparisons of 5’-flanking sequences of two Xenopus laevis Rax and one X. tropicalis
Rax, identified the conserved noncoding sequence (CNS1) that is located 2 kb upstream of
Rax gene. Transgenic assay and luciferase assay using X. laevis embryos showed that
CNS1 has cis-regulatory activity of Rax. Further sequence comparisons revealed that CNS1
is conserved among many vertebrates including human, and that CNS1 contains perfectly
conserved consensus binding sites for OTX and SOX proteins. As the Otx2 and Sox2 mRNA
are coexpressed with Rax mRNA in the eye priomordia, we hypothesized that Otx2 and Sox2
proteins directly bind to CNS1 to regulate Rax expression. Electrophoretic mobility shift
assays and chromatin immunoprecipitation assays revealed the direct bindings of Otx2 and
Sox2 proteins to CNS1. In addition, reporter assays using frog animal cap cells and human
cultured cells (HEK293T) showed that Otx2 and Sox2 proteins activate Rax transcription in
an interdependent manner. Moreover, we showed that Otx2 and Sox2 proteins interact with
each other in vitro and in vivo. These results demonstrate direct linkages among three
transcription factor encoding genes associated with eye development. Currently, we are
attempting to expand the transcriptional network identified in this study by comparative
genomics approach for the understanding of brain development.


Temporal and spatial expression pattern of FGF ligands and receptors during
Xenopus development
Robert Lea, Enrique Amaya and Karel Dorey
The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, UK.
Fibroblast growth factor (FGF) signalling plays a major role during early vertebrate
development. It is involved in the specification of the mesoderm, the control of
morphogenetic movements, the patterning of the anterior-posterior axis and in neural
induction.
        In mammals, 22 FGF ligands have been identified which can be grouped into seven
subfamilies according to their sequence homology and functional data All FGFs ligands but
fgf17 and fgf18 have been identified in the Xenopus tropicalis genome by synteny and
sequence homology. We have cloned 17 fgf ligands so far and have analysed their temporal
expression by RT-PCR and spatial expression by whole mount in situ hybridisation.
        The data presented here constitute the most complete description of the temporal
and spatial expression pattern of FGF ligands and receptors during vertebrate development
to date. We hope that it will help us to understand better the pleiotropic roles of FGF
signalling during development.

Carbonic Anhydrase XII (CA12) is required for ciliated cell development
in the epidermis of Xenopus tropicalis

Eamon Dubaissi and Nancy Papalopulu, Faculty of Life Sciences,Michael Smith Building
University of Manchester,Oxford Road, Manchester, M13 9PT, UK

In Xenopus embryos, the ciliated cells of the epidermis develop by a two-step mechanism of
differentiation in the inner layer of the epidermis, followed by a process of intercalation into
the outer layer, where they undergo ciliogenesis. Little is known about how this process is
regulated and indeed what the role of other intercalating cells (INCs) is. CA12 is a
membrane bound enzyme with its catalytic site facing the external environment. It is known
to catalyse the hydration of carbon dioxide to bicarbonate, which releases protons and
modifies the local pH. In the current study we show that CA12 is expressed in INCs,
adjacent to ciliated cells. We also show that knocking down CA12 leads to cilia dysfunction
and the results suggest that this is due to loss of ciliated cells. If this is the case, it could be
that the INCs act as support cells for the ciliated cells during their development and could
illustrate a general principle of how pH changes can regulate developmental processes.



   In vivo information with in vitro tools

   David Du Pasquier1, Anthony Sébillot1, Sophie Pallud-Mothré1, Andrew Tindall1,
Stéphanie Mateos1, Grégory Lemkine1 and Barbara Demeneix1,2

1: Watchfrog S.A, 1 Rue Pierre Fontaine, Bâtiment Geneavenir 3, 91000 Evry, France
2: UMR CNRS 5166 Evolution des Régulations Endocriniennes, Dept Régulations,
Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle
7 rue Cuvier, 75005 Paris, France

   WatchFrog creates and markets innovative solutions for the in vivo detection of a wide
range of chemical and pharmaceutical substances. Basically, WatchFrog models will “light
up” (through emission of fluorescence) when a biological function is activated.
   Small model organisms derived from amphibians exploit the physiological pertinence of
whole organism approaches with the advantages of accelerated testing in either micro-
volumes (96 well plates) or flow through readings. Many environmental biomarkers could be
good candidates to mimic the potential effects of emerging contaminants on vertebrate
physiology. For example, we have developed models to detect hormonal disruption of thyroid
functions. Fluorescent X. laevis tadpoles bearing genetic constructs integrating hormonal
responsive elements are used for physiological-based screens for potential endocrine
signaling disruptors.
   The genetic and physiological proximity between humans and Xenopus is particularly well
established and recognized for endocrine regulation for which Xenopus is OECD reference
model. Other physiological and pathological fields can also benefit from small model
organisms technology such as developing alternative rapid in vivo test for chronic toxicity in
vertebrates. For these, Xenopus is again relevant in that it very rapidly develops a metabolic
capacity, which can be predictive of human metabolism including liver and kidney functions.


The XMeis3 Transcription Factor Acts Downstream of Canonical Wnt-Signaling to
Regulate Multi-Cell-Fate Decisions in the Developing Xenopus Nervous System
Yaniv Elkouby and Dale Frank, Department of Biochemistry, Rappaport Institute for
Research in the Medical Sciences, Technion Medical School, Haifa, Israel.

In Xenopus and other vertebrates, canonical Wnt-signaling plays a key role in specifying
posterior cell fates, such as hindbrain, neural crest, and primary neurons. In Xenopus
embryos, inhibition of canonical Wnt-signaling or knock down of XMeis3 transcription factor
protein activity leads to the loss of these posterior nervous system cell fates. The activation
of XMeis3 gene expression in the embryo requires canonical Wnt-signaling; XMeis3
expression is not activated in dkk-1 injected or Xwnt3a morphant embryos. The XMeis3
transcription factor induces these posterior neural cell fates, by acting downstream to the
canonical Wnt-pathway. The loss of canonical Wnt-signaling, by either ectopic expression of
the dkk-1 protein or Xwnt3A morpholino knockdown leads to a loss of hindbrain, neural crest
and primary neuron cell type phenotype, similar to the XMeis3 morphant phenotype. Ectopic
XMeis3 protein expression in the dkk-1 and Xwnt3a morphant embryos rescues phenotypes
at both a morphological and cell marker expression level. The reciprocal effect is not
observed; ectopic Xwnt3a protein expression does not rescue XMeis3 morphant embryo
phenotypes. Our experiments in explants suggest that Xwnt3a protein expressed in the
gastrula-stage dorsal-lateral mesoderm region is secreted to overlying neuralized ectoderm
where it induces XMeis3 expression and the subsequent formation of these different
posterior neural cell types. Thus, the activation of Xwnt3a expression in the mesoderm
induces XMeis3 protein expression in the neural plate, which subsequently acts to caudalize
neuralized cells to posterior cell fates.



Serum- and glucocorticoid-inducible kinase 1 (SGK1) promotes cell survival by
suppressing the expression of death-inducing signaling complex (DISC) components
in early Xenopus development
Tatsuya Endo, Morioh Kusakabe, Kazunori Sunadome, Takuya Yamamoto and Eisuke
Nishida
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto
University, Japan

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is an evolutionarily conserved
serine/threonine kinase. Although SGK1 has been shown to be involved in various biological
events, its role in early development remains unknown. Here we analyze the function of the
Xenopus ortholog of SGK1 (xSGK1) in early Xenopus embryos. Whole mount in situ
hybridization reveals that xSGK1 expression is seen in the vegetal hemisphere at the
blastula stage, in the endoderm and the dorsal mesoderm at the gastrula stages, and in the
endoderm at the neurula stage. At the tailbud stage, the expression is observed in the pineal
gland and the dorsal fin. Microinjection of xSGK1 antisense morpholino oligonucleotides
(MOs) into the dorsal region results in a shortened body axis, with defective eye(s) and
reduced head structures. Microinjection of MOs into the ventral region results in a defective
tail. TUNEL assays show that xSGK1 knockdown causes excessive apoptosis at the late
gastrula and neurula stages. Our microarray analysis indicates that the expression levels of
TRADD, FADD and caspase-10, which constitute a death-inducing signaling complex
(DISC), are up-regulated in xSGK1 knockdown embryos. Moreover, we show that xSGK1-
MOs-induced apoptosis is inhibited by co-expression of a dominant negative form of FADD.
These results suggest that xSGK1 plays an essential role in cell survival during early
embryonic development, at least in part, by suppressing the expression of these pro-
apoptotic genes.




Screening for FGF targets: Functional Analysis of the miRNA processing protein Lin-
28 in Xenopus tropicalis
Laura Faas, Julie Affleck and Harry V. Isaacs. Biology Department, University of York,
Heslington, York, YO10 5YW, UK

The heterochronic gene lin-28 encodes a highly conserved RNA binding protein which was
originally identified as a gene involved in coordinating the relative timing of key
developmental events in C.elegans. It has also recently been identified as one of the four
factors sufficient for reprogramming human somatic stem cells, along with Oct4, Nanog and
Sox2. In an Affymetrix microarray based screen for targets of the FGF signaling pathway
using we have recently identified lin-28 as an FGF-dependent gene. In situ hybridization
analysis shows that lin-28 is expressed from early gastrula stage around the blastopore,
consistent with the early FGF expression pattern. Down-regulation of lin-28 via antisense
morpholino (MO) injections resulted in severe axis abnormalities, including shortened trunk
with small or missing tail, and severely reduced head and eyes. This phenotype can be
rescued by co-injection of the MO+ lin-28mRNA. Histological analysis of the morphants
showed reduced brain vesicles and severely impaired eye development, as well as structural
disorganization of somatic muscle and other tissues. Recent work in embryonic stem cells
indicates the role of lin-28 on regulating the processing of precursors of the let-7 miRNA
family members, key developmental regulator factors that control gene expression by
posttranscriptional repression. Our Northern blot analysis of the lin-28 morphants reveals up-
regulation of the mature form of the let-7 miRNA. The effects of let-7 over-expression in the
context of lin-28 expression are discussed.


Anterior-posterior tissue positional values and anterior-posterior cell polarity maintain
the axis of cell intercalation during Xenopus convergent extension.

Caroline Flournoy, Ray Keller: Department of Biology at the University of Virginia,
Charlottesville, Virginia 22904 USA.

The convergent extension of the dorsal mesoderm of Xenopus is driven by a stereotyped
and coordinated mediolateral intercalation of cells to produce a narrower, longer tissue.
Recent work in flies and frog demonstrates that cells use a local disparity in anterior-posterior
positional value as a cue that orients the axis of cell intercalation perpendicular to the
anterior-posterior body axis, thereby assuring that the resulting extension occurs along the
anterior posterior axis. We show that post-involution dorsal notochordal and somitic
mesoderm retains sensitivity to disparities in anterior-posterior tissue identity and can
reorient the axis cell intercalation accordingly through early development. Cells of explanted,
anti-parallel, apposed notochord and somitic mesoderm reorient their mediolaterally-
elongated axes to maintain an orientation perpendicular to the largest local disparity in
anterior-posterior tissue identity. The reoriented cells intercalate efficiently to drive extension
perpendicular to the axis of intercalation. The timing of the reorientation response varies with
developmental stage but can occur in as little as 20 minutes after tissues from disparate
anterior-posterior levels are apposed. The direction of reorientation is always such that the
anterior sides of the cells turn toward the most anterior tissue positional value. Positional
value-mediated reorientation occurs within notochordal and somitic tissue and between the
two tissues, indicating that a common signaling system underlies this phenomenon in both
tissues. Our results indicate that orientation of cell intercalation with respect to the tissue
anterior-posterior axis is dynamically maintained, even as successive rounds of intercalation
diminish the anterior-posterior identity differential between neighboring cells.

Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) affects TGF-β signaling
and is necessary for Xenopus development.
Mary Fortner, Tuzer Kalkan, Gerald Thomsen

          TRAF4 is one of six Tumor Necrosis Factor Receptor-Associated Factor (TRAF)
family members and the only one that does not function in the Tumor Necrosis Factor (TNF)
or Interluekin/Toll-related (IL/TLR) signaling pathways. The overexpression of TRAF4 in
Xenopus animal caps results in an increase in both BMP and nodal signaling, whereas,
knockdown of TRAF4 reduces BMP and nodal signaling. During neurula stages, TRAF4 is
expressed in the neural plate and neural crest. Blocking TRAF4 disrupts the formation of
neural crest, indicating that it plays a role in neural crest specification. In addition to the
expression in the neural plate and neural crest, TRAF4 is expressed in the mesoderm during
gastrulation. In Xenopus laevis, there are two paralogs of TRAF4 that encode proteins that
are 95% identical. The knockdown of each paralog exhibits different effects on mesoderm
during gastrulation. Blocking TRAF4a shows a very mild phenotype of a slightly shortened
body axis and reduced head size. However, blocking TRAF4b shows a massive reduction of
brachyury as well as loss of anterior and axial structures. We will present new findings on
the effects of the knockdown of TRAF4 in mesoderm.

Annotation of Xenopus Proteins in UniProtKB/Swiss-Prot.

Rebecca E. Foulger, Rolf Apweiler and the UniProt Consortium.

EMBL Outstation - Hinxton, European Bioinformatics Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom.

Sequencing of the Xenopus tropicalis genome, together with the growing number of Xenopus
EST/cDNA projects, is generating a vast amount of nucleotide sequence data. Studying the
encoded protein sequences is a key step in interpreting the Xenopus genomes. UniProt
(Universal Protein Resource; http://www.uniprot.org) provides a central resource on protein
sequences and functional annotation, and contains the manually annotated
UniProtKB/Swiss-Prot section and the automatically annotated UniProtKB/TrEMBL section.

Xenopus curation in UniProtKB/Swiss-Prot focuses on X. laevis and X .tropicalis, with priority
given to proteins with mammalian homologs. For each protein, sequences and experimental
data are collated and summarised using controlled vocabularies, including gene ontology
(GO) terms, and free text. The GO database is just one of more than 60 databases cross-
referenced in UniProtKB, and we are currently working on generating links to XenBase.

UniProt data is freely available and fortnightly releases are provided in Fasta, flat file and
XML formats. With the ongoing curation of Xenopus protein sequences, UniProt aims to
support the Xenopus research community. We are happy to provide demonstrations of
UniProtKB, and welcome feedback regarding which Xenopus proteins or molecular pathways
are of particular interest to you.

A TRANSGENIC NON-INVASIVE CASPASE DETECTION SYSTEM REPORTING
APOPTOSIS AND DIFFERENTIATION DURING NORMAL XENOPUS DEVELOPMENT
FRANSEN Mathias1, WILLEMARCK Nicolas, VLEMINCKX Kris1
1
 Unit of Developmental Biology, Department for Molecular Biomedical Research, VIB-Ghent
University, Ghent, Belgium.
E-mail: Mathias.Fransen@dmbr.UGent.be

Apoptosis plays an essential role in animal development and homeostasis. Disorders of this
process cause various pathologies, including autoimmune and neurodegenerative diseases.
Caspases are central mediators of the apoptotic process, but there is growing evidence that
these cysteine proteases also have apoptosis-independent functions, e.g. in the
differentiation of lens fiber cells and erythrocytes.

Because of functional redundancy and the static nature of current apoptotic assays, the
developmental role of caspases is presumably greatly underestimated. We have designed
and evaluated new non-invasive reporter systems that register caspase activation during
apoptotic and differentiation events in living Xenopus embryos. These transgenic reporter
systems are based on the generation of a fluorescent signal that is dependent on the activity
of specific caspases. Because of their external development and transparency, Xenopus
embryos are ideal for real time fluorescent analysis.

During early development specific and highly dynamic patterns of caspase activity are
detected (e.g. in the brain, spinal cord, eye, kidneys) in the transgenic reporter lines.
Interestingly, regional differences can be seen between caspase-3 (C3) and caspase-9 (C9)
activity (e.g. in the embryonic blood islands). High caspase activity is also detected in the
eyes in individual cells in all retina layers (including the photoreceptors) and in the
developing lens at the onset of differentiation of lens fiber cells. Overlap with TUNEL-positive
cells proves the ability to detect apoptotic cells. To evaluate the developmental role of
different caspases during early development, loss-of-function experiments are performed
with specific morpholinos. Depletion of C3 did not induce major changes in the fluorescent
patterns of the C3-reporter line, pointing to functional redundancy, e.g. with C7. Indeed,
combined C3-C7 MO injections were detrimental for the developing embryo suggesting an
important role of those caspases during early development.

We will further use the offspring of the transgenic caspase reporter lines for hypothesis-
driven experiments and to identify new roles of caspases during selected developmental
processes.




Eomesodermin and Nodal signaling coregulate Xenopus zygotic VegT expression in
mesoderm formation.
Masakazu Fukuda1, Shuji Takahashi1, Yoshikazu Haramoto2, Yasuko Onuma1, Chang-Yeol
Yeo3, Shoichi Ishiura1 and Makoto Asashima1,2,4
1
  The University of Tokyo, Japan. 2ICORP Organ Regeneration Project, Japan Science and
Technology Agency (JST), Japan. 3Ewha Women's University, Korea. 4National Institute of
Advanced Industrial Sciences and Technology (AIST), Japan.

In Xenopus embryogenesis, the T-box gene VegT plays a crucial role during mesendoderm
specification. In particular, the maternal VegT, which functions as a germ cell determinant,
has been extensively investigated. However, the functional and transcriptional programs of
zygotic VegT (zVegT) are still unknown. By using comparative genomics approach and
translation-blocking experiments by the antisense morpholino oligo (MO) of zVegT, we
demonstrated that zVegT is the orthologous gene of zebrafish Spadetail, which is essential
for posterior mesoderm developmet, and functions in the proper paraxial mesoderm
formation. Additionally, transgenic techniques were identified two upstream that are
necessary and sufficient to induce mesoderm-specific expression of zVegT. Each sequences
contained putative binding sites of transcription factor for Nodal signaling mediator Forkhead
box H1 (FoxH1) and T-box proteins. Electrophoretic mobility shift assays showed that FoxH1
and T-box proteins bound to the cis-regulatory sequence, respectively. Furthermore, the
splice-inhibiting MO of T-box gene Eomesodermin (Eomes), which is regulated by Nodal
signaling, led to suppress the endogenous expression of zVegT. Based on these data, we
conclude that Eomes and Nodal/FoxH1 signaling directly coregulate zVegT gene expression
in mesoderm and that zVegT is associated with the gene network of paraxial mesoderm
formation.

Myofibrillogenesis mutants in Xenopus tropicalis.
Timothy J. Geach, Antia Abu-Daya, Michael J. Reilly & Lyle B. Zimmerman
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA. UK. Tel.
+44 20 8816 2469. e-mail. tgeach@nimr.mrc.ac.uk.

Xenopus tropicalis is proving extremely amenable for use in forward genetic screens and
mapping. A recent chemical mutagenesis screen for developmental mutations has identified
a panel of interesting phenotypes in skeletal and cardiac muscle development. These
mutants are paralysed and show defective cardiac rhythm scored by visual observation,
polarized light birefringence, phalloidin and antibody studies. We have mapped one of these
mutants, mrs lot (mlo), to a 4 cM interval on chromosome 6 that contains titin, a large
structural sarcomeric protein. Additional mutants, petrified, bert huggins and sophronia do
not complement mlo suggesting they are also mutant alleles of the same gene. An additional
mutant, dicky ticker (dit), displays an identical phenotype to, but genetically complements,
the mlo mutation. Indeed, dit has been mapped to a 0.7cM interval on chromosome 2
making it a distinct mutant from mlo. This interval contains 14 genes. Of these, at least
three (hspB1/hsp27, a mitochondrial malate dehydrogenase homolog mdh2 and unc-45b)
are expressed in skeletal and cardiac muscle in developing Xenopus embryos. We are
continuing to refine our analysis with genetic, expression, and sequencing studies to identify
the responsible locus.

Characterization of RCSD1 during embryogenesis of Xenopus laevis

Susanne Gessert, Tata Purushothama Rao, Ioan Ovidiu Sirbu and Michael Kühl

Institute for Biochemistry and Molecular Biology, University of Ulm, Albert-Einstein-Allee 11,
D-89081 Ulm

Duboraya has recently been shown to be important for zebrafish left-right asymmetry via
non-canonical Wnt signalling. In human, the Duboraya orthologue CapZIP (CapZ interacting
protein) is a key player in modulating the actin cytoskeleton during stress. In this study, we
have identified the Duboraya othologue in Xenopus laevis and mouse, named RCSD1. In
both organisms, several splice variants could be detected by RT-PCR and sequencing. In
mouse, RCSD1 is expressed in the developing heart as well as in neural tissue and paraxial
mesoderm. In Xenopus, the first heart-specific expression can be detected at stage 20 in the
cardiac progenitor cell pool with a preference in prospective first heart field cells suggesting a
heterogeneity of this progenitor population already at this stage. Loss-of-function analysis
using a specific antisense morpholino oligonucleotide revealed that RCSD1 is required for
normal heart development in Xenopus. Unilateral downregulation of RCSD1 led to a reduced
expression of heart marker genes in the first heart field at stage 29, whereas early heart
specification at stage 20 is not affected. These data suggest that regulation of the actin
cytoskeleton does not only influence cell morphology and morphogenetic movements but
also gene expression in cardiac progenitor cells.




Functional characterization of DMRT5 in forebrain and olfactory placode development
Moers V. Ghogomu S., Bellefroid EJ .
Laboratoire d'Embryologie Moléculaire, Université Libre de Bruxelles, Institut de Biologie et
de Médecine Moléculaires (IBMM), Gosselies, Belgium.

Vertebrate DM domain genes encode a large family of proteins related to the Drosophila
doublesex (Dsx) and Caenorhabditis elegans mab-3 transcription factors involved in sexual
development. Here we show that the Xenopus DMRT5 gene is expressed at early neurula
stage in the anterior neural plate and that it is later restricted to the dorsal telencephalon,
ventral diencephalon and olfactory placodes. A similar highly restricted expression profile has
been observed in the chicken and mouse embryos. In gel shift assays, XDMRT5 exhibits
similar DNA-binding activities as Dsx and mab3. In a yeast two-hybrid assay, we identified
AES (amino-terminal enhancer of split) as a XDMRT5 putative interacting partner. Together,
our data indicate that DMRT5 may play an important role in forebrain and olfactory placode
development and suggest that its activity may be regulated by the recruitment of AES and
the related Groucho protein. Overexpression and knockdown experiments in Xenopus are
currently underway to approach its function.


The role of Alsin, a Guanine Exchange Factor for Rab5 GTPase, in early Xenopus
development
Penny Gill and Nancy Papalopulu, Faculty of Life Sciences, Michael Smith Building
University of Manchester, Oxford Road, Manchester, M13 9PT, UK

Mutations within the ALS2 gene, which encodes Alsin, causes a number of recessive motor
neuron diseases (MND), such as amyotrophic lateral sclerosis (ALS) , primary lateral
sclerosis and hereditary spastic paraplegia. In early Xenopus development Alsin is
expressed throughout the ectoderm which consists of two cell populations, superficial
polarised epithelial cells and deep, non-epithelial cells. Alsin is a guanine exchange factor
(GEF) for Rab5 GTPase which is involved in endosome/membrane trafficking and endosome
fusion in cells. The role of Alsin has not yet been investigated in polarised cells or in Xenopus
embryogenesis. During development Alsin is maternally expressed; zygotic expression
levels peak at stage 18. After cloning the full-length Xenopus Alsin, the aim is to determine
its function by over-expression, knock-down, dominant negative and constitutively active
forms. A specific aim is to determine whether Alsin is involved in the establishment in cell
polarity via the endocytosis pathway in early blastomeres.



Microarray study of Xrx1-driven gene regulation in the Xenopus laevis eye field
Guido Giudetti, Daniele Biasci, Martina Giannaccini, Chiara Capriotti, Giuseppina Barsacchi,
Massimiliano Andreazzoli - University of Pisa, Department of biology, Cell and
Developmental Biology Unit

The paired-like Xrx1 gene was cloned more than a decade ago, and functional studies have
shown that this transcription factor is involved in eye field specification and retinal stem cell
maintenance, thus being crucial for eye development. Still, little is known about the
interacting partners or downstream targets of this gene, thus hampering a comprehensive
knowledge of its actual role and activities in gene expression regulation during initial and late
phases of eye development.
To shed more light on the gene network regulated by Xrx1 activity, we designed a high-
throughput screen to identify Xrx1 regulated genes by use of Affymetrix microarrays.
Overexpression of Xrx1 was induced by injection of synthetic mRNA into animal dorsal
blastomeres, while functional knock-down was obtained by use of morpholino
oligonucleotides. The gene expression profile of injected embryos was compared with the
expression profile of non-injected embryos at stage 13, leading to the identification of 793
differentially expressed transcripts in the gain-of-function experimental setup and to 1893
differentially expressed transcripts in the loss-of-function setup. Notably, a subset of 121
transcripts results differentially expressed in both experimental conditions.
By means of an extensive bioinformatic effort to remap Affymetrix probeset sequences to the
latest UniGene database release, we obtained up to date annotations for all the transcripts
available on the microarray, being also able to estimate the quantity of non-annotated and/or
non-matching transcripts.
Real-time PCR validation, functional analysis based on updated transcript mapping and GO
annotation, as well as systematic in situ hybridizations for relevant non-annotated
sequences, lead to initial corroboration of microarray data and to the identification of possible
novel activities of Xrx1 during eye field specification and retina development.


The role of the transactivation domain CCR2 of the LIM homeodomain protein Xlim-1
in the Spemann-Mangold organizer
Y. GOYA1, K. MISAWA1, T. FUKUSHIMA2, S. TAKAHASHI2, M. TAIRA1
(1Dept. of Biol. Sci., Grad. Sch. of Sci.;, 2 Grad. Sch. of Agric.& Life Sci., Univ. of Tokyo)

Xlim-1 is the LIM homeodomain transcription factor expressed in the organizer and activates
several organizer genes. Therefore, study of the regulatory mechanism of Xlim-1 activity at
the protein level helps us to understand the intricate transcriptional networks in the organizer.
We have previously reported that Xlim-1 requires conserved five tyrosines (5Y) in the C-
terminal conserved region 2 (CCR2) to initiate secondary axis formation, suggesting the
possibility that a coactivator binds to CCR2 in a 5Y-dependent manner. In this study, we
further investigated the molecular and biological roles of the 5Y motif in CCR2. We first
examined which Xlim-1 target genes depend on the 5Y motif for their gene expression. The
C-terminal region of Xlim-1 (Ct) and its 5YA mutant, in which 5Ys were replaced with 5As,
were overexpressed in the organizer region so as to deplete a putative 5Y binding
coactivator, called p YBC. We found that Ct downregulated the expression of gsc and chd
in a 5Y-dependent manner and this effect was especially remarkable for chd. Furthermore,
although Xlim-1 and its cofactors Ldb1 and Ssbp3a upregulated gsc, otx2, and chd in animal
caps, the 5YA mutant of Xlim-1 in this context greatly reduced its activity specifically for chd
expression. This result suggests that each target gene requires different levels of the
p YBC and the most FYB -dependent gene is chd. To identify the p YBC, pulldown
analysis using GST-CCR2 and GST-CCR2(5YA) was performed with egg and embryo
extracts. By SDS-PAGE and mass spectrometry, we identified several proteins which may
bind to CCR2. We are now analyzing these proteins in detail.



Lef/Tcf subtype specifity in early Xenopus development

Dietmar Gradl, Stephanie van Venrooy, Doris Wedlich

Department of Molecular Developmental and Cell Physiology, Institute of Zoology II,
University of Karlsruhe
The four members of the Lef/Tcf transcription factor family (Lef-1, Tcf-1, Tcf-3 and Tcf-4) are
the main nuclear transducer of Wnt/β-catenin signaling. Lef/Tcfs share similar mechanisms
of target gene regulation, including DNA binding to a consensus target site 5´-
G/CTTTGA/TA/T-3´ and competition of groucho and b-catenin for Lef/Tcf binding. They are
expressed in overlapping regions, with some expression domains specific for a particular
family member, including XTcf-4 expression in the midbrain and XLef-1 expression at the
midbrain/hindbrain boundary. Furthermore, we show that Lef/Tcfs regulate their own
expression and the expression of their family members in a complex manner. However, in
early development they appear to play non-redundant roles: Knock-down of XTcf-4 results in
midbrain defects, knock-down of XLef-1 disturbs convergent extension movements and
knock-down of XTcf-3 leads to neural plate defects.
In order to identify Tcf-subtype specific target genes responsible for these non-redundant
roles we subtracted the transcriptome of XTcf-4 depleted neuralized animal caps from XTcf-3
depleted ones. We identified the Cold-Inducible RNA binding protein CIRP as XTcf-3 specific
target. Knock-down of XCIRP and XTcf-3 result in a similar phenotype: a broadening of
anterior neural tissue. We provide evidence, that a general stabilization of mRNA by the
XTcf-3 target XCIRP is important for lateral constriction of the anterior CNS.




Methylation regulates the activity of the transcription factor ILF3 in Xenopus embryos.
Ophelie Cazanove, Julie Batut1, Garry Scarlett, Katie Mumford, Stuart Elgar, Sarah Thresh,
Isabelle Neant1, Marc Moreau1 and Matthew Guille. Institute of Biomedical and Biomolecular
Sciences, School of Biological Sciences, University of Portsmouth, Portsmouth, U.K. 1.
Centre de Biologie du Developpement, Groupment de Recherche 2688, Unite Mixte de
Recherche 5547, Centre National de la Recherche Scientifique, 118 Route de Narbonne,
Toulouse, France.

Modification of proteins by methylation has emerged as a key regulatory mechanism in many
cellular processes including gene control. 80-90% of the arginine methylation in the cell is
performed by the protein arginine methyl transferase PRMT1. ILF3, a protein involved in
gene regulation at several levels, has been shown to be a substrate and regulator of PRMT1
in mammals. Here we show that the Xenopus orthologue of ILF3 (Xilf3) is methylated in vivo,
and at least in vitro this methylation is by Xprmt1b. The in vitro methylation of Xilf3 inhibits its
ability to bind to DNA whilst leaving RNA binding activity unaltered. Consistent with these
activities having a role in vivo the DNA binding activity of the Xilf3 containing CBTF complex
and the transcription of its target gene, Xgata2, is altered in response to overexpression of
Xprmt1b. However, in contrast to other RNA binding proteins, changing methylation does not
alter the sub-cellular localization of Xilf3. Several other proteins involved in gene regulation
can bind both RNA and DNA; these data demonstrate a mechanism by which such binding
activities may be controlled independently.


Early organizer-specific retinoic acid signaling is required for head formation
Michal Gur, *Christof Niehrs and Abraham Fainsod
Dept. of Cellular Biochemistry and Human Genetics, Hebrew University-Hadassah Medical
School, POB 12272, Jerusalem 91120, Israel. * Division of Molecular Embryology,
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany.

Retinoic acid (RA) plays numerous important signaling roles during embryogenesis.
Deviations from normal RA concentrations, result in severe teratogenic effects in embryonic
structures, including craniofacial malformations. In Xenopus embryos, treatment with both
excess and low levels of RA lead to defective anterior head structures. In transgenic
embryos RA signaling becomes active at the onset of gastrulation localized to Spemann’s
organizer. This localized activity suggests an early role for RA in the “organizer” function. We
detect RA signaling in induced organizers, using the secondary axis assay. Based on the
head phenotypes induced by RA loss-of-function we hypothesized that head formation
requires early RA signaling. Induction of head-containing secondary axes concomitant with
inhibition of RA signaling resulted in less efficient head formation. Localized reduction of RA
levels in the endogenous organizer resulted in a reduction of head markers. The requirement
for RA during head formation was mapped to the early gastrula stage, corresponding to the
activity of RA in Spemann’s organizer. The effects of manipulating RA levels in the embryo
suggest a dual affect for this signaling pathway. RA loss-of-function down regulates the
expression of several organizer genes, while treatment with high levels of RA leads to the
same results. Titration of RA levels revealed a double threshold in response to RA signaling,
where RA signaling is required for gene expression, but high levels of RA inhibit their
expression. These observations suggest that in addition to its known negative regulatory role
during neurula, RA is required early in the formation of anterior head regions. This novel
activity of RA signaling on head formation is in agreement with the proposed reduction of this
signal in DiGeorge/Velocardiofacial, Vitamin A Deficiency and Fetal Alcohol syndromes and
the craniofacial malformations they exhibit.

Xenopus tropicalis Mutation Resource Informatics
Samantha Carruthers1, Amanda Hall1, Carlos Torroja1, Richard Harland2, Lyle Zimmerman3,
Derek Stemple1.
1. The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
2. The University of California, Berkeley, USA.
3. The National Institute for Medical Research, Mill Hill, London, UK.

We have established methods to screen libraries of randomly mutagenised X. tropicalis in
order to find knockouts in genes of interest by exon re-sequencing. We are in the process of
generating a library of 6000 mutant X. tropicalis in order to recover stop mutations in 175
genes over five years. This screen requires complex informatics support and we have
designed a database to track the genes being screened, record mutations and upload them
to Ensembl. Mutant animals are recorded in a database that keeps track of founders,
offspring and observed phenotypes. Furthermore an external website has been set up in
order for the community to request that their genes of interest are screened for potential
mutations and there is a tracking page to observe the progress of the request,
http://www.sanger.ac.uk/Projects/X_tropicalis/tilling/.

Molecular characterization of Ptf1a function during neurogenesis.
Marie Hedderich, Tomas Pieler and Kristine Henningfeld.
Department of Developmental Biochemistry, University of Goettingen, Justus-von-Liebig
Weg 11, 37077 Goettingen, Germany.

The bHLH transcription factor Ptf1a/p48 is essential for the specification of a pancreatic fate
and later is required to drive expression of the exocrine digestive enzymes. More recently, in
the retina, cerebellum and dorsal spinal cord, Ptf1a has been shown to play a central role in
the generation of GABAergic inhibitory neurons at the expense of excitatory glutamatergic
neurons. At the molecular level, Ptf1a forms a heterotrimeric complex by direct interaction
with a common bHLH E-protein and a member of the Su(H) family.
In Xenopus, we found that Ptf1a, similar to the bHLH neuronal determination factor X-ngnr-1,
strongly induced neurogenesis. However, Ptf1a and X-ngnr-1 induced distinct neuronal
subtypes. X-ngnr-1 strongly activated the expression of XHox11L2/Tlx3, which marks a
subset of sensory neurons and is a selector gene determining glutamatergic over GABAergic
cell fates. In contrast, Ptf1a induced the expression of Pax2 and glutamic acid decarboxylase
(Gad1a) expression, markers for GABAergic interneurons and inhibited XHox11L2
expression. To better understand the shared and unique activities of Ptf1a and X-ngnr-1, we
performed a comparative temporal analysis of genes induced by both transcription factors.
Ptf1a and X-ngnr-1 were found to induce neurogenesis through the activation of the same
proneural factors in a similar temporal cascade and later act to drive the expression of
unique neuronal subtype markers. Through the use of chimeric constructs, we found that
neuronal identity of X-ngnr-1 is localized to the bHLH domain, but not that of Ptf1a.
Correspondingly, overexpression of Ptf1a bearing mutations in the C-terminus that disrupt
interaction with Su(H), did not influence the ability to drive neurogenesis. These mutations,
however, disrupted the ability of Ptf1a to confer the correct neuronal cell-type identity.


Wnt8 stimulates TCF3 phosphorylation to relieve transcriptional repression and
promote ventro-posterior development
Hiroki Hikasa and Sergei Y. Sokol
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine,
New York, NY 10029, USA.

Wnt signaling pathways play a major role in the control of cell proliferation, cell polarity and
cell fate determination in multiple developmental processes. A commonly accepted
mechanism of Wnt signaling involves the stabilization of beta-catenin, which forms a complex
with TCF family members leading to target gene activation. Here we show that Wnt8
signaling leads to phosphorylation of TCF3 in the ventral marginal zone and relieves TCF3-
dependent transcriptional repression of the Vent2 gene, which has a pivotal role in early
patterning. This process requires homeodomain-interacting protein kinase 2 (HIPK2), which
interacts with and phosphorylates TCF3. Consistent with these findings, Xenopus embryos
depleted of endogenous HIPK2 reveal enhanced dorso-anterior development and decreased
Vent2 expression, whereas TCF3-depleted embryos upregulate Vent2 and develop
microcephaly. Our results emphasize the significance of alternative Wnt signaling
mechanisms operating in a vertebrate embryo. Moreover, we discuss the interaction
between Wnt8/beta-catenin pathway and Wnt8/HIPK2 pathway.


Functional differences between dorsal and ventral pancreas specific genes
Lori Dawn Horb1, Zeina Jarikji1, Farhana Shariff1, Craig A. Mandato2, Ken W.Y. Cho3 and
Marko E. Horb1,2
1
  Laboratory of Molecular Organogenesis, Institut de Recherches Cliniques de Montréal,
Canada
2
  Department of Anatomy and Cell Biology, McGill University, Canada
3
  Developmental Biology Center and the Department of Developmental and Cell Biology,
University of California Irvine, USA


The mature pancreas is derived from separate pancreatic buds that arise from spatially
distinct regions- the dorsal pancreas from just below the notochord and the ventral pancreas
adjacent to the liver. Several lines of evidence have revealed that each bud is induced by
different signaling pathways, but less is known about the molecular signals that act
downstream of these initial inductive interactions. To identify molecular differences between
these two buds we isolated individual dorsal and ventral pancreatic buds from stage 38/39
Xenopus laevis tadpoles, prior to fusion, and screened the Xenopus Affymetrix Genechip.
Our hypothesis was that the dorsal pancreas would be enriched in genes involved in
endocrine cell differentiation, and the ventral pancreas in genes involved in exocrine
pancreas development. As a result of this screen, we identified over 200 genes as enriched
greater than 2 fold in either the dorsal or ventral pancreas. In agreement with our hypothesis
we found endocrine-specific genes to be enriched in the dorsal pancreas, whereas genes
involved in acinar cell differentiation and pancreas morphogenesis were enriched in the
ventral pancreas. We selected four candidate genes, 2 dorsal and 2 ventral and examined
their function in early pancreas development. Knockdown of either dorsal gene inhibited
endocrine cell differentiation to differing degrees, whereas knockdown of either ventral gene
resulted in perturbed fusion of the dorsal and ventral buds and loss of acinar cells, but had
no effect on pancreatic endocrine cell differentiation. These results are the first to examine
the molecular differences between the dorsal and ventral pancreas and demonstrate that
ventral genes direct development of acinar cells and pancreatic bud fusion, while dorsal
genes function in endocrine cell fate specification.


Role of a localized TRIM protein in Xenopus axis formation.
Douglas W. Houston and Tawny Cuykendall.
The University of Iowa, Department of Biology. 257 BB, Iowa City, IA 52242, USA

The activation of Wnt/beta-catenin signaling on the future dorsal side of the blastula is
necessary and sufficient for axis formation in Xenopus and other vertebrates. Wnt signaling
is initiated by dorsal enrichment of vegetally localized molecules following rotation of the egg
cortex after fertilization. Both localized wnt11 mRNA and protein inhibitors of beta-catenin
degradation, Dishevelled and GBP, have been implicated, but the mechanisms activating
Wnt signaling in axis formation still remain elusive. Because vegetally localized RNAs are
important for this process, we have conducted a microarray screen to identify novel mRNAs
localized to the vegetal cortex. We present evidence that a localized mRNA encoding a
Tripartite Motif Protein (Trim), Trim36, plays a critical role in Xenopus axis formation.
Maternal antisense inhibition of trim36 resulted in ventralized embryos, with reductions in
dorsal beta-catenin accumulation and Wnt target gene expression. We further present
experiments to identify the extent that Trim36 interacts with Wnt/beta-catenin signaling and
cortical rotation mechanisms.


Robust stability of the embryonic dorsal axial pattern requires ONT1, a secreted
scaffold for Chordin degradation
Hidehiko Inomata, Tomoko Haraguchi and Yoshiki Sasai

Dorsal axial formation during vertebrate embryogenesis exhibits robust resistance to
perturbations in patterning signals. However, how such stability is supported at the molecular
level remains largely elusive. Here we show that Xenopus ONT1, an Olfactomedin-class
secreted protein, stabilizes dorsal axial formation by restricting Chordin activity on the dorsal
side. When ONT1 function is attenuated, the embryo becomes hyper-dorsalized by a
normally sub-effective dose of Chordin. ONT1 binds Chordin and BMP1/Tolloid-class
proteinases (B1TP) via distinct domains, and acts as a secreted scaffold that enhances
B1TP-mediated Chordin degradation by facilitating the enzyme-substrate association. ONT1
is indispensable for fine-tuning BMP signaling in the dorsal axial tissue, and a similar role has
been suggested for dorsally expressed BMPs, such as ADMP. Simultaneous inhibition of
ONT1 and dorsally expressed BMPs (ADMP and BMP2) synergistically caused drastic
dorsalization. We discuss a dual-safety model for dorsal axis formation, consisting of
compensatory regulatory pathways involving ONT1/B1TP and dorsally expressed BMPs.


Genetic Studies of Inner Ear Development in Xenopus tropicalis
Holly Ironfield, Lyle Zimmerman
National Institute for Medical Research, Medical Research Council

Xenopus tropicalis provides an excellent model system for studying the formation of the inner
ear. The otic placode is easily identified and the transparency of the later embryo allows the
inner ear to be observed throughout its development. A pilot chemical mutagenesis screen
has revealed seven mutations affecting inner ear formation. These were identified due to
defective otolith formation and abnormalities in the size of the otic vesicle both resulting in
balance defects.

To identify the genes responsible for these phenotypes, we are pursuing genetic maping
strategies incorporating candidate gene approaches, genome scanning, and gynogenetic
linkage to centromere markers. The mutations are being characterised phenotypically using
wholemount in situ hybridisation with molecular markers for otic placode induction, and
patterning. Differentiating structures such as the statioacoustic ganglion and hair cellsa re
being visualized by confocal microscopy using fluorescen antibody staining . We hope that
studying these mutations will yield information on novel mechanisms in vertebrate inner ear
development.

Analysis of the fibroblast growth factor dependent transcriptome
Harry V Isaacs, Peter Branney, Laura Faas, Mary Elizabeth Pownall
Department of Biology University of York, York YO10 5YW, United Kingdom.

Fibroblast growth factor (FGF) signaling has key roles in germ layer specification in the early
vertebrate embryo. We have undertaken an Affymetrix microarray based screen in order to
identify effectors of FGF signalling required for germ layer specification in Xenopus
gastrulation. We have identified early targets of FGF signalling in the embryo by examining
effects on gene expression resulting from FGF inhibition shortly after the initial activation of
zygotic FGF signaling in the late blastula. Data comparing the effects on gene expression
resulting from inhibition of FGF signaling with dominant negative versions of FGF receptor 1
and FGF receptor 4 will be presented. We have further investigated signaling and
transcriptional targets downstream of individual FGF receptors using mutant versions of
FGFR1, FGFR2, FGFR3 and FGFR4 that can be activated by drug induced
homodimerization during gastrula stages. These combined approaches have allowed us to
identify many novel, putative effectors of FGF signaling. Knockdown studies in Xenopus
tropicalis indicate roles for many of these genes in the processes of germ layer specification
and tissue morphogenesis downstream of FGF signaling. Analysis of gene expression in
FGF target knockdown embryos will allow the mapping of FGF dependent regulatory
pathways in the amphibian embryo. Results from these ongoing studies will be presented.

A W-linked DM-W gene may be a sex (ovary)-determining gene in Xenopus laevis.
Michihiko Ito, Shin Yoshimoto, Tadayoshi Shiba, Nobuhiko Takamatsu
Dept. Biosci., Sch. Sci., Kitasato Univ. JAPAN

The sexual fate of metazoans is determined genetically or by environmental factors. In the
former case, heterogametic sex chromosomes determine the male (XY♂) or female (ZW♀)
fate in many species of vertebrates. In the XX/XY sex-determining system, the Y-linked SRY
genes of most mammals and the DMY/DMRT1Y genes of the teleost fish medaka have been
characterized as sex-determining genes that trigger formation of the testis. However, the
molecular mechanism of the ZZ/ZW-type system in vertebrates including the clawed frog
Xenopus laevis is unknown. Here we isolated X. laevis female genome-specific DM domain
gene, DM-W and supplied the first molecular evidence indicating the existence of a W-
chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high
identity with that of DMRT1, but DM-W had no significant sequence similarity to the
transactivation domain of DMRT1. In non-mammalian vertebrates, DMRT1 is involved in
testis formation. DMRT1 or DM-W was expressed exclusively in the primordial gonads of
both ZZ and ZW or ZW tadpoles, respectively. We next produced transgenic tadpoles
carrying a DM-W expression or knockdown vector. Some developing gonads of the ZZ
transgenic tadpoles carrying the expression vector showed ovarian cavities and primary
oocytes. Importantly, two ZW frogs bearing the knockdown vector developed testicular
gonads. Taken together, these results strongly suggest that DM-W is a sex (ovary)-
determining gene. Now we propose a novel model for ZZ/ZW-type sex determination in
which DM-W determines the development of bipotential gonads into ovaries by interfering
with the testis formation induced by DMRT1.

IGFBP-4 is a canonical Wnt inhibitor that promotes cardiac myogenesis
Yuzuru Ito1, Weidong Zhu2, Ichiro Shiojima2, Issei Komuro2, Makoto Asashima3,4,5
1 Center for Structuring Life Sciences, Graduate School of Arts and Sciences, The University
of Tokyo
2 Department of Cardiovascular Science and Medicine, Chiba University Graduate School of
Medicine
3 ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST)
4 Department of Life Sciences (Biology), Graduate School of Arts and Science, The
University of Tokyo
5 National Institute of Advanced Industrial Sciences and Technology (AIST)

In amphibians, cardiogenesis is initiated by the combined action of Wnt antagonists from the
Spemann-organizer and several endodermal factors on adjacent mesoderm; however, the
mechanisms by which the induced precardiac anlage forms a beating and separated heart
remain unclear. Here we report a previously unknown function for IGFBP-4 as a cardiogenic
growth factor during late tailbud stage. IGFBP-4 is member of Insulin-like growth factor
binding proteins (IGFBPs), binding to and modulating the actions of insulin-like growth
factors (IGFs). Although some of the actions of IGFBPs have been reported to be
independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are
largely unknown. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of
IGFBP-4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of
IGFBP-4 was independent of its IGF binding activity but was mediated by the inhibitory effect
on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8
(Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and
inhibited Wnt3A binding to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect
of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. Thus, IGFBP-4 is a
canonical Wnt inhibitor that promotes cardiac myogenesis, and provides a molecular link
between IGF signaling and Wnt signaling.

The sperm-surface glycoprotein, SGP, is necessary for fertilization of the frog,
Xenopus laevis
Iwao, Y. 1, Nagai, K.1, Ishida, T.1, Hashimoto, T.1, Harada, Y.1, Ueno, S.1, Ueda, Y.1, and
Kubo, H.2
1
  Lab. Mol. Dev. Biol., Yamaguchi Univ., Japan. 2 Dept. Med. Biol. Tokyo Metro. Inst. Med.
Sci., Japan

  To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a
monoclonal antibody against a sperm-surface glycoprotein (SGP) on the sperm surface was
obtained by immunizing mice with a membrane fraction of the sperm of the frog, Xenopus
laevis, followed by screening of the culture supernatants based on their inhibitory activity
against fertilization. The fertilization of both jellied and denuded eggs was effectively
inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment,
indicating that the antibody recognizes the molecule on sperm surface necessary for
fertilization. On Western blots, anti-SGP antibody recognized larger molecules with molecular
mass of 65 - 150 kDa and minor smaller molecules with 20 - 28 kDa in the sperm membrane
vesicles. Periodate oxidation of SGP completely abolished the antigenicity, indicating the
involvement of the carbohydrate moieties of SGP in fertilization. SGP was distributed on
almost whole sperm surface, probably as an integral membrane protein in close association
with microfilaments in a lipid/membrane raft fraction. Larger amount of membrane vesicles
containing SGP bound to the surface of the animal than the vegetal hemisphere in
unfertilized eggs, whereas such binding was not observed in fertilized eggs. These results
indicate that SGP mediates sperm-egg membrane binding and is responsible for
establishment of fertilization in Xenopus .

Requirement of keratin-related protein Ouroboros as immune antigens for
metamorphic tail regression in Xenopus
Yumi Izutsu1, Mitsugu Maeno1, Masanori Taira2, Kazunori Onoe3
1. Department of Biology, Faculty of Science, Niigata University; 2. Department of Biological
Sciences, Graduate School of Science, University of Tokyo; 3. Institute for Genetic Medicine,
Hokkaido University, Japan.

From our previous finding, where a skin graft from the tadpole tail was rejected by a
syngeneic young adult of Xenopus J strain, we proposed a hypothesis that newly
differentiated adult-type T cells induce tail absorption during metamorphosis by recognizing
tail skin cells as non-self. We have obtained antisera from adult frogs immunized by
repeated transplantations with the same strain of larval tail skin grafts, which specifically
recognize “adult T cell responding antigens”. Using the antisera, we identified two keratin-
related proteins, named Ouroboros1 and Ouroboros2. These were expressed specifically in
the tail skin at metamorphic climax stages in coordinate fashion, and the recombinant
proteins derived from these genes generated T cell responses of adult Xenopus in vitro. We
generated ouro1 and ouro2 double transgenic animals using a nuclear transplantation
technique to analyze functions of ouro genes in vivo. When the two ouro genes were
overexpressed in the tail region, precocious tail degeneration occurred accompanied with T
cell accumulation. Then, we generated first filial (F1) generation using a strong ouro
expressing-transgenic female frog mated with a normal wild-type male. Among the F1
tadpoles, 75% of tadpoles exhibited the phenotypes. In contrast, no degeneration was seen
in sibling tadpoles at stage 52 before adult-type T cell had fully differentiated. Conversely,
when expression of the ouro genes was knocked-down, the tail skin tissue was retained in
part even after metamorphosis. These results indicate that the ouro gene products
participate in the process of tail regression as immune antigens.


Characterization of XHNF1ß and XHNF6 in context of pancreas development in
Xenopus laevis

Christine Jäckh and Tomas Pieler
Department of Developmental Biochemistry, GZMB, University of Göttingen



Abstract:
We have previously reported that retinoic acid signalling is required for the formation of the
dorsal pancreas as early as during gastrulation. Furthermore, we could also show that the
combined expression of the transcription factors Pdx1/Xlhbox8 and Ptf1a/p48 in the
endoderm is sufficient to induce ectopic pancreatic differentiation from the gut epithelium.
Our current work aims at a better understanding of the events that occur downstream of RA
signalling and upstream of the transcriptional activation of Pdx1/Xlhbox8 and Ptf1a/p48 in
the context of pancreas development. Knockout studies in the mouse had identified two
candidate upstream regulators of these two genes, namely HNF1ß/TCF2 and HNF6/onecut-
1. On these grounds, we have isolated HNF6 from Xenopus laevis and analyzed its
expression. Effects on pancreas development generated by gain- and loss of function
approaches for both candidate regulators, HNF1ß and HNF6, have been analyzed.
Furthermore, we are also investigating a possible link between RA signalling during
gastrulation and HNF1ß expression in the early endoderm.
Bioinformatics Identifies Cis-Regulatory Elements in Retinoic Acid-Responsive Genes
of the Xenopus Embryo
Amanda Janesick, Bruce Blumberg
Department of Developmental and Cell Biology, University of California, Irvine

Retinoid receptors are nuclear proteins that respond to signals from lipid-soluble ligands to
regulate gene transcription and expression. The precise regulation of retinoic acid (RA)
levels is critically important to the temporal and spatial expression of genes involved in
vertebrate development. Retinoid-responsive genes can be regulated directly (the immediate
early genes) or indirectly (the late response genes) by RA. Deciphering the regulatory
networks that connect one gene to another in the retinoic acid receptor (RAR) signaling
cascade remains a significant challenge. We address this by examining three distinct groups
of retinoid-responsive genes using bioinformatics: genes expressed in pre-placodal ectoderm
(a region that possesses the correct signals to become sensory organs), genes belonging to
the same synexpression group, and immediate early genes downstream of RARs. We
assume that noncoding sequence of these genes hold enough information to provide insight
to whether the genes are regulated directly or indirectly by retinoic acid, and what
transcription factors interact with them. Unlike previous studies that examine a few kilobases
of noncoding sequence, our approach has been to examine the entire gene, comprising 20
kb upstream of the translation initiation site, the coding sequence, and 20 kb downstream of
the stop codon. To our knowledge, comparable studies have not been performed in any
organism. Identifying regulatory elements in a eukaryotic organism in silico is a powerful
approach to focus and inform subsequent wet lab techniques (e.g., ChIP-CHIP) that seek to
accomplish the same goal. The results of this study will facilitate the elucidation of the gene
regulatory networks under the control of RA signaling. Moreover, the combination of
bioinformatic and experimental approaches will allow us to concentrate on conserved
promoter elements in the target genes and lead to rapid progress in the defining regulatory
hierarchies downstream of RAR.

Identification and characterization of new canonical Wnt target genes in Xenopus

Sylvie Janssens1, Tinneke Denayer1, Tom Deroo1, Steffen Durinck2, Pieter De Bleser3,
Dominique Vlieghe3, Yves Moreau2, Frans Van Roy1 and Kris Vleminckx1.
1
  Department for Molecular Biomedical Research, VIB, Ghent, Belgium
2
  SCD-ESAT, KULeuven, Kasteelpark Arenberg 10, 3001, Leuven-Heverlee, Belgium
3
  Bioinformatics Core, Department of Molecular Biomedical Research, VIB, Ghent, Belgium

The canonical Wnt pathway has a pivotal role during animal development and in adult life. Its
activation leads at its nuclear endpoint to the transcriptional activation of specific target
genes. In many different species and systems putative Wnt target genes have been
identified. Still, only a limited number are verified to be directly regulated by the pathway.
Since they represent potential therapeutic anchor points for the treatment of cancer or for
stem cell therapies, there is a tremendous need for the identification of new primary Wnt
target genes that are only induced in a particular cellular context. For this they are ideally
identified in the complex context of a whole embryo, which provides tissue- and organ-
specificity, and with an inducible system, which favors the identification of true primary
targets. Hence, we designed and optimized inducible transgenic Wnt pathway interfering
(activating or repressing) systems acting at the nuclear endpoint of the pathway and
performed microarray analysis on neurula stage Xenopus embryos. Interestingly this
generated a list of less than 100 genes which were affected by manipulating the Wnt
pathway. Remarkably, 21% of these were known Wnt target genes. Several others were
genes associated with processes where the canonical Wnt pathway is known to be involved
at the stage analysed (e.g. anterior-posterior patterning and differentiation) further validating
our system. The majority of these genes was also identified by a learning algorithm that
identifies potential Wnt target genes using an in silico genomic screen. Further analysis of
  these genes by in situ hybridization showed that the majority are not ectopically induced by
  the Wnt activating construct even though this is ubiquitously expresses. This indicates that
  the genomic and cellular context imposes specificity on the induced genes. We are now
  exploiting this remarkable feature to identify novel organ-specific Wnt target genes.



  Left-right patterning in X. laevis: xPitx3 and the retinoic acid shield
  Jerant, L., Smoczer, C., Wolanski, M., Khosrow Shahian, F., Crawford, M.J.
  Dept. of Biological Sciences, University of Windsor, 401 Sunset, Windsor, Ontario, N9B 3P4

  There is a relationship between laterality and somitogenesis processes that has been
  observed in mutant embryos where the retinoic acid pathway has been compromised1.
  Laterality gives rise to left- and right-specific information and is responsible for the
  asymmetries seen in the viscera of the body. The development of somites along the
  anterior-posterior axis must be coordinated in a bilaterally symmetric fashion for normal
  phenotype to be obtained in the skeletal muscles of the trunk. These two processes occur in
  adjacent tissues; presomitic mesoderm and lateral plate mesoderm, and yet utilize some of
  the same genes to result in opposite patterns. It has been suggested that a buffering effect
  by retinoic acid (RA) allows for the processes to be unaffected by one another and occur
  simultaneously in development1, 2. In our lab, the knockdown and over-expression of the
  transcription factor xPitx3, affects both the anterior-posterior and left-right body axes. We
  have formulated the hypothesis that xPitx3 is acting upstream of RA, possibly in multiple
  pathways during development. From a microarray experiment, we have obtained genes that
  are involved in the RA pathway and contain multiple putative xPitx3 binding sites. It has also
  been shown in the midbrain, that treatment with RA can rescue the deleterious effects that
  Pitx3 mutations can have on dopaminergic neurons3. Defects in the looping of the heart and
  gut, leading to various forms of heterotaxy, spike our curiosity about the role of xPitx3 in the
  laterality pathway as well. The work presented to date explores the relationship between
  xPitx3 and genes both in the laterality cascade, as well as genes possibly regulating the
  retinoic acid shield, allowing the somitogenesis clock to tick unaffected by the left-right axis
  being established.

1. Vermot, J. & Pourquie, O. (2005) Nature. 435, 215-20.
2. Kawakami, Y., et al. (2005) Nature. 435, 165-71.
3. Smidt, M.P., et al. (2007) Development. 134, 2673-2684.




  Paraxial Protocadherin is required for proper otic vesicle formation
  Barbara Jung1, Alexandra Schambony2, Doris Wedlich1
  1
    Zoological Institute II, Department of Molecular Developmental- and Cellphysiology,
  University of Karlsruhe, Germany; 2Biology Department, Developmental Biology, Friedrich-
  Alexander-University Erlangen-Nuremberg, Germany

  The entire inner ear, including its innervation, develops from an ectodermal thickening, the
  otic placode. During its development, morphogenesis and patterning are controlled by a
  number of signaling pathways including Fgf, canonical and noncanonical Wnt signaling.
  Xenopus Paraxial Protocadherin (PAPC) participates in noncanonical Wnt signaling during
  gastrulation movements (Unterseher et al., 2004; Medina et al., 2004) and its expression is
  regulated by the novel Wnt5a/Ror2 pathway (Schambony and Wedlich, 2007). During otic
development XPAPC arises very early in the presumptive otic placode (st. 17) and is
expressed throughout the otic development at least until st. 45 (Hu et al., 2006).
We show that XPAPC is mainly present in the lateral epithelium in the early otocyst.
First loss-of-function experiments using antisense-Morpholino indicate that loss of XPAPC
impairs otic vesicle formation. The most severe alterations can be observed during early
development at the invagination process. We show that the absence of XPAPC leads to
early morphological failures but does not affect the expression of different patterning genes
and the apical-basal epithelial structure.


Novel repressor of Notch signaling regulates patterning of left-right axis
Daisuke Sakano, Akiko Kato and Yoichi Kato
Department of Biomedical Sciences, Florida State University College of Medicine

   The Notch signaling pathway is an evolutionary conserved intracellular signaling pathway
and regulates downstream responses, such as cell-fate specification, progenitor cell
maintenance, boundary formation, cell proliferation and apoptosis. Since it still remains
incomplete to understand the regulatory mechanism of transcription for Notch signaling, we
performed co-immunoprecipitation combined with proteomics analysis to find novel
transcriptional regulators for Notch signaling. We identified BCL6, a transcriptional
repressor, as a novel Notch-associated factor. BCL6 is expressed in ectodermal and
mesodermal tissues at early developmental stages and its expression is observed in the
nervous system, eye, and somite at later stages. We next performed knockdown
experiments of BCL6 by using Morpholino Oligonucleotides in Xenopus embryos. The
depletion of BCL6 leads to the disorientation of gut origin, gut coiling and heart, and the
suppression of left-side specific gene expression, Pitx2 but not Xnr1, in the left lateral plate
mesoderm (LPM), suggesting that BCL6 is involved in patterning of left-right (LR) axis. We
have also confirmed that Notch receptor is required for patterning of LR axis and the
expression of Xnr1 and Pitx2 in the left LPM of Xenopus embryos similar to other
vertebrates. Interestingly, the overexpression of active form of Notch receptor suppressed
the expression of Pitx2 but not Xnr1 in the left LPM and this Notch activity was inhibited by
co-expression of BCL6 and BCL6 co-repressor (BCOR). Taken together, our data indicate
that BCL6 inhibits the expression of some downstream targets of Notch signaling, which
could suppress the expression of Pitx2 in the left LPM, to maintain the expression of Pitx2 in
patterning of LR axis.

Translational regulation of axis formation
Mark W. Kennedy, Phillip G. Andrews and Kenneth R. Kao. Terry Fox Cancer Labs, Faculty
of Medicine, Memorial University, St. John’s, NL, Canada

        Formation of the primary axis during embryonic development is directed by maternal
factors present at fertilization. However, it is not clear why genes that define the axis are not
expressed until late blastula stages despite the presence of maternal mRNAs that encode
proteins which directly regulate their expression. Xenopus laevis embryos employ multiple
mechanisms to regulate the post-transcriptional levels of mRNA and proteins during
oogenesis and embryogenesis. These range from the regulation of mRNA stability through
the 3’UTR by polyadenylation (or deadenylation) or miRNA activity, the regulation of
translation mediated by RNA binding proteins and also the regulation of protein activity by
inhibitory complexes. We report the identification, embryonic expression and regulation of the
Xenopus orthologue of the β-catenin binding and regulating protein, Bcl9. XBcl9 mRNA
levels are highest and uniformly distributed but XBcl9 protein is barely detectable in pre-MBT
embryos. Interestingly, XBcl9 protein levels increase after MBT as mRNA levels decrease
and is found in dorsal nuclei at stages 8-8.5. Analysis of the XBcl9 cDNA revealed a novel
mRNA sequence motif present in the 5’UTR that was required for inhibition of expression of
XBcl9 protein prior to the MBT. Furthermore, the timing of the alleviation of translational
repression of XBcl9 mRNA is consistent with the timing of accumulation of XBcl9 protein in
dorsal nuclei. These results suggest a novel mechanism, regulated by the temporally
restricted translation of XBcl9, which contributes to asymmetric gene expression in the early
Xenopus embryo.




Mapping the grinch mutation using half-tetrad analysis
Dipankan Bhattacharya1, Richard M. Harland1, Mustafa K. Khokha2
1
  MCB – UC-Berkeley 2Pediatrics/Genetics – Yale University School of Medicine

We have identified numerous recessive mutations in X. tropicalis and efforts are now being
made to quickly and efficiently map the mutations. One of the first mutations we identified is
grinch, which is believed to be a defect in the lymphatic or circulatory system. Starting
around stage 30 – 35, mutants develop ventral edema and swelling near the kidney or lymph
heart. In order to map this, and other recessive mutations, we have found gynogenesis and
half-tetrad analysis to be a very efficient technique. In a previous effort, we identified
centromeric markers for the 10 linkage groups of X. tropicalis. Using these centromeric
markers, we found that grinch was linked to LG 10 (linkage in 82/84 half-tetrads). Additional
mapping using half-tetrad analysis shows that the mutation is near the centromere, and to
date, we have mapped the grinch mutation to an interval of 3 cM on LG 10. We show that
half-tetrad analysis can be used to map mutations first to one of the 10 chromosomes and
then to narrow regions of the genome.

Novel function for Ryk in the positive control of noncanonical Wnt signaling by
modulating βarr2-dependent endocytosis during Xenopus gastrulation

Gun-Hwa Kim, Jung Hyun Her, and Jin-Kwan Han1
1
 Department of Life Science, Division of Molecular and Life Sciences, Pohang University of
Science and Technology, San 31, Hyoja Dong, Pohang, Kyungbuk, 790-784, Republic of
Korea

Single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions
as a Wnt receptor. However, Ryk’s correlation with Wnt/Fz signaling is poorly understood.
Here, we report that Ryk regulates Xenopus convergent extension (CE) movements via the
β-arrestin 2 (βarr2)-dependent endocytic process triggered by noncanonical Wnt signaling.
During Xenopus gastrulation, βarr2-mediated endocytosis of Frizzled 7 (Fz7) and dishevelled
(Dvl/Dsh) actually occurs in the dorsal marginal zone (DMZ) tissues, which actively
participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of
Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and βarr2,
cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the
noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11
prevents Dsh endocytosis in DMZ tissues. Our study suggests that Ryk functions as an
essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of
Xenopus CE movements.

Xenopus Wntless and Retromer complex cooperate to regulate XWnt4 secretion
Hyunjoon Kim and Jin-Kwan Han
Division of Molecular and Life Sciences, Pohang University of Science and Technology, San
31, Hyoja- dong, Pohang, Kyungbuk, 790-784, Republic of Korea

Wnt signaling is implicated in a variety of developmental and pathological processes. The
molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated.
Wntless, an evolutionarily conserved multi-pass transmembrane protein, is a dedicated
secretion factor of Wnt proteins that participates in Drosophila embryogenesis. In this study,
we show that Xenopus Wntless (XWntless) regulates the secretion of a specific Wnt ligand,
XWnt4, and that this regulation is specifically required for eye development in Xenopus
laevis. Moreover, the Retromer complex is required for XWntless recycling to regulate
XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino
(MO) resulted in various Wnt deficiency phenotypes affecting mesoderm induction,
gastrulation cell movements, neural induction, neural tube closure, and eye development.
Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects, but not
other deficiencies. These results collectively suggest that XWntless and the Retromer
complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye
development.

The Roles of XFrsX, an FRS-like docking Protein, in Developing Xenopus Embryos
Seulki Kim1, Yeon-Jin Kim1, Yong Hwan Kim2, Jeung-Kyun Kim2, Won-Sun Kim2 and Chang-
Yeol Yeo1
1
  Department of Life Science and Division of Life and Pharmaceutical Sciences, Ewha
Womans University, Seoul 120-750, Korea
2
  Department of Life Science, Sogang University, Seoul 121-742, Korea

FGF signaling plays pivotal roles for mesoderm induction and axis formation during early
embryogenesis of Xenopus laevis. Frs2 (FGF receptor substrate 2) has been previously
identified as a mediator that links the activation of FGF receptor to its downstream effectors.
In this study, we identified an XFrs-like protein, Xenopus FrsX (XFrsX), and investigated its
role during embryogenesis. XFrsX is expressed maternally and zygotically with specific
expression patterns throughout the early development. XFrsX is expressed around the
blastopore in gastrula and in neural crest cells, head and somites in tailbud stage embryos.
Knockdown of XFrsX function caused defects in the formation of eye and head, particularly
the expression of eye marker genes. Our results suggest that XFrsX plays an important role
in anterior neural induction during early embryogenesis.


XPOFUT1 regulates Nodal signaling during early development of Xenopus laevis
Yeon-Jin Kim, Yong Hwan Kim and Chang-Yeol Yeo
Department of Life Science and Division of Life and Pharmaceutical Sciences, Ewha
Womans University, Seoul 120-750, Korea

Nodal signaling plays pivotal roles for mesoderm induction and axis formation during early
embryogenesis of Xenopus laevis. It has been previously reported that EGF-CFC factor, an
essential cofactor of Nodal signaling, is O-fucosylated at the conserved EGF-like motif in
CHO cells. In mouse and Drosophila, the conserved EGF-like motives of Notch proteins are
O-fucosylated by POFUT1. We isolated an ortholog of POFUT1 from Xenopus laevis and
named it XPOFUT1. XPOFUT1 is expressed maternally and zygotically through the early
development. XPOFUT1 is expressed ubiquitously in blastula and around the blastopore lip
in gastrula. In tail-bud stage embryos, XPOFUT1 is expressed in the optic placode, optic
vesicle, brain, somites, brachial arch, pronephric tubule and pronephric duct. Knockdown of
XPOFUT1 function caused defects in axis formation, somitogenesis, and the formation of
eye and melanocytes. XPOFUT1 knockdown also reduced the activation of an activin-
responsive element and Smad2 phosphorylation in late blastula, and reduced the expression
of Nodal target genes while it did not influence activin-induced gene expression. We also
found that XPOFUT1 interacts with the Cripto. Our results suggest that XPOFUT1 plays
important roles during early embryogenesis, at least in part, by regulating Nodal signaling
possibly through the post-translational modification of EGF-CFC factors.

Role of Notch signaling in mesoderm formation of Xenopus laevis embryo
Yuki Haruta1, Chiharu Sakamoto1, Susumu Imaoka1, and Tsutomu Kinoshita1,2
1
  Department of Bioscience, School of Science and Technology, Kwansei Gakuin University
2
  Department of Life Science, Faculty of Science, Rikkyo University

Notch signaling pathway is widely conserved from vertebrate to invertebrate and mediates
the specification of numerous cell fates during developmental processes. In Xenopus laevis,
the role of Notch signaling has been studied in detail in neurogenesis, whereas Notch
signaling in gastrula embryo remains unknown. In Xenopus gastrula embryo, Xdelta1, one
of the Notch ligands, is expressed in the prospective mesoderm prior to Xbra expression.
However gene expression of Notch target gene, XESR1, is not detected during gastrulation.
Here we have examined the role of another HES family gene, XESR5, in order to understand
the role of Xdelta1 during gastrulation. It has been shown that XESR5 is involved in the
segmentation of paraxial mesoderm under the control of Notch signaling. RT-PCR and
whole-mount in situ hybridization showed that XESR5 is expressed predominantly in the
marginal zone of the early gastrula embryo. However, animal cap assay indicated that gene
expression of XESR5 is induced not by Notch signaling, but by nodal signaling. In order to
clarify the role of XESR5 in the marginal zone of gastrula embryos, dominant-negative form
of XESR5 was injected into the prospective mesoderm. The truncated form of XESR5
caused the ectopic expression of XESR1, which resulted in the decrease of Xbra expression
and the defective gastrulation. These results suggest that XESR5 produces the competent
area for the mesoderm formation by suppressing the gene expression of XESR1.
Morpholino oligo DNA of Xdelta1 caused severe gastrulation defect in the injection site,
showing that Notch signaling is necessary for mesoderm formation. Taking together, these
results suggest that Notch signaling in Xenopus gastrula embryo plays an essential role for
mesoderm formation in HES -independent manner.


GTPBP2 is a positive regulator of TGF-β signaling, and is required for mesodermal
patterning.
Arif Kirmizitas, Gerald H. Thomsen
Department of Biochemistry and Cell Biology, Center for Developmental Genetics, Stony
Brook University

        The Transforming Growth Factor β (TGF-β) superfamily of signaling proteins regulate
a diverse set of biological processes, including cell proliferation, adhesion, migration,
apoptosis, differentiation and embryonic pattern formation. Because of its key role in these
processes, a plethora of regulators are evolved to modulate TGF-β signaling. The TGF-β
superfamily is comprised of about 30 ligands, which are commonly grouped into two broad
sub-families based on their downstream signaling effectors: the TGF-β/Nodal/Activin and
BMP/GDF families. To identify new molecules that regulate the intracellular BMP/Smad1
signaling pathway, we have undertaken a yeast two-hybrid screen using Smad1, and
retrieved GTPBP2 as a binding partner for Smad1. GTPBP2 and its close homolog GTPBP1
are large GTPases of unknown function. Here, we introduce GTPBP2 as a novel component
of TGF-β /Smad signaling. GTPBP2 interacts with a subset of Smad proteins, and consistent
with these interactions, GTPBP2 induces mesoderm in animal cap explants and enhances
canonical TGF-β signaling pathways in Xenopus and HepG2 cells. GTPBP2 mRNA is
maternal and expressed in a dynamic pattern in developing Xenopus embryos. By knocking
down GTPBP2 levels, we showed that GTPBP2 is required for mesodermal patterning, and
BMP signaling. Finally, GTPBP2 is a nuclear protein and colocalizes to nuclear foci with
Smad1.

Regulation of Cadherin-11 gene expression in Xenopus laevis

Katja Weißbach, Susanne Hasenauer and Almut Köhler
Department of Molecular Developmental and Cell Physiology, Institute of Zoology II,
University of Karlsruhe

Cadherins are cell-cell adhesion molecules important for tissue cohesion, cell separation and
cell migration during development. The classical type II Cadherin-11 is mainly expressed in
migrating cranial and trunk neural crest cells of Xenopus laevis.
We isolated a -2070 bp cadherin-11 promoter fragment and analyzed the putative binding
motifs. The reporter gene EGFP was activated in a tissue specific manner by the -2070
fragment. While a shorter fragment of -1170 bp was not active in a luciferase assay the full
fragment could drive reporter gene expression. The expression could be enhanced by the
neural crest inductive molecule c-myc and was repressed by AP-2, an early neural crest
specific marker.
It was shown by in situ hybridization and RT-PCR that c-myc could activate Cad-11
expression. In contrast, injection of Cad-11 morpholino resulted in increased c-myc levels
and increased apoptosis on the expense of neural markers while expression of neural crest
markers was delayed. This indicates that Cad-11 keeps cells in an early neural crest fate and
delays the further specification.
Inhibition of the canonical wnt/β-catenin pathway by injection of TCF3 morpholino
oligonucleotides resulted in a reduction of Cad-11 expression.
These results indicate that the -2070 bp promoter fragment is able to drive tissue specific
expression. The expression is regulated by neural crest specific transcription factors. The
canonical wnt/β-catenin signalling pathway interacts with Cad-11 expression leaving the
question whether this is due to a direct function on Cad-11 as target gene of wnt/β-catenin
signalling or an influence of an increased cytoplasmic β-catenin pool on Cad-11.

Regulation of XTcf-4 in the developing midbrain

Sabrina F. Koenig, Dietmar Gradl, Doris Wedlich

Department of Molecular Developmental and Cell Physiology, Institute of Zoology II,
University of Karlsruhe

XTcf-4 as a nuclear transducer of the Wnt/β-catenin signaling cascade shows a distinct
expression as a narrow stripe in the midbrain, which first appears at neurula stage (st.16)
embryos. The expression of XTcf-4 is regulated via canonical wnt signals in the midbrain.
Here we demonstrate that this autoregulatory loop depends on a functional Lef/Tcf
consensus sequence on the XTcf-4 promoter. Chromatin IP (ChIP) experiments revealed
that in Xenopus tissue culture (XTC) cells this site is endogenously occupied by β-catenin.
In a transgenic approach we can show that a 3.85 kb promoter fragment is mainly active in
the developing CNS, including the XTcf-4 expression field. High promoter activity in the
forebrain and retina indicate that additional regulatory elements are necessary to drive the
correct temporal and spatial XTcf-4 expression. However, upon microinjection the 3,85 kb
promoter fragment mimics endogenous XTcf-4 expression, while a shorter construct of 2,8
kb does not. Thus, relevant information for the spatial expression of XTcf-4 is located
upstream of position -2800. Therefore we are analyzing the functionalities of putative binding
sites on the XTcf-4 promoter, including En-2 (at positions -3840 and -2880), Pax-5 (-3240)
and p53 (-3620). In reporter gene assays we can show that en-2 and pax-5 regulate XTcf-4
activity via their binding sites on the XTcf-4 promoter. On the other hand, our finding that
XTcf-4 is necessary for en-2 expression indicates that a complex regulatory network of
transcription factors acts in concert for the embryonic midbrain patterning.

XFz7/PAPC mediated signalling regulates gene transcription in gastrula embryos

Isabelle Köster and Herbert Steinbeisser
Institute of Human Genetics, Department of Developmental Genetics, University Heidelberg,
INF 366, 69120 Heidelberg

During Xenopus gastrulation, the involuting mesoderm gets into contact with the inner layer
of the blastocoel roof, but the two tissues remain separated. Key molecules for tissue
separation are Paraxial Protocadherin (XPAPC) and the Xenopus Frizzled-7-receptor (Xfz7),
which contribute to non-canonical Wnt-signalling and activate Rho, JNK and PKC.
To determine whether PAPC and Xfz7 also play a role in regulating the transcription of target
genes, microarray analysis was performed on the Agilent Xenopus oligo microarray system.
In animal cap tissue ectopically expressing PAPC and Fz7, we found 56 upregulated and 58
downregulated genes. The array results were confirmed for a subset of these genes
by RT-PCR, qRT-PCR and “whole mount” in situ-hybridizations.
Two components of GTPase mediated signalling, a novel RhoGAP and a novel ArfGAP,
which are under negative regulation of PAPC and Fz7, are further investigated. Gain and
loss of function studies for ArfGAP and RhoGAP indicate that these proteins contribute to the
regulation of convergent extension and tissue separation.


Expression and functional analysis of XASPP2 during Xenopus neural development
Qinghua Kong, Shuhua Zhao, Yingjie Xia and Bingyu Mao
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

The apoptosis stimulating proteins of p53 (ASPP) family proteins play important roles in the
control of apoptosis and cell growth. ASPP2 is a binding partner of p53 and acts as a positive
regulator of p53 mediated apoptosis. The ASPP2-null mice exhibit developmental defects
predominantly in the central nervous system and heart. However, its exact role during early
embryonic development is still unclear. Here, we have cloned the Xenopus ASPP2
(XASPP2) and studied its expression and functions in early development. XASPP2 shares
78% identity with mouse ASPP2. RT-PCR analysis indicates that it is maternally expressed
and can be detected throughout embryogenesis. By in situ hybridization, it is detected
strongly in the animal pole during early cleavage and blastula stages. At neurula stage it is
specifically expressed in the neural plate, while at tailbud stage it is predominantly detected
in the brain, eye and cement gland. Knock-down of ASPP2 by specific morpholino leads to
head and eye defects in the injected side. Immunostaining of phosphorylated histone H3
shows a decrease of mitotic cells in the injected area. These data suggests that XASPP2 is
likely involved in anterior neural development through regulation of cell proliferation.

Xwnt11 influences the subcellular localization of XPAPC during Xenopus gastrulation
Bianca Kraft1, Alexandra Schambony2 and Doris Wedlich1
1
  Zoological Institute II, Department of Molecular Developmental- and Cellphysiology,
University of Karlsruhe, Germany; 2Biology Department, Developmental Biology, Friedrich-
Alexander-University Erlangen-Nuremberg, Germany

During gastrulation, convergent extension (CE) movements shape the dorsal anterior-
posterior body axis. These specialized cell movements require strict coordination of cell
polarity and cell migration including reorganization of the cytoskeleton and modulation of cell-
cell adhesion. The non-canonical Wnt/PCP pathway predominantly controls CE movments.
We have previously shown that Xenopus paraxial protocadherin (XPAPC) is a key molecule
in coordinating CE movements by activating RhoA and JNK (Unterseher et al., 2004).
XPAPC is localized in the medial and lateral membranes of the bipolar mesodermal cells and
regulates their parallel alignment along the mediolateral axis. Since Wnt/PCP controls
polarity formation in mesodermal cells (Wallingford et al., 2000), we address the question
whether this pathway regulates the subcellular XPAPC localization.
First results in Keller tissue explants show that XPAPC localization is influenced by Xwnt11,
but not by Xwnt5a. Time-lapse microscopy analysis reveals that XPAPC containing vesicles
are dynamically transported in retrograde and anterograde direction. To distinguish between
different intracellular traffic processes that participate in XPAPC localization and turn-over we
use the Xenopus cell lines A6 and XTC as model systems. Here, we demonstrate that the
average speed in retrograde transport is about 1,5 µm/s while the anterograde speed is
about 0,7 µm/s. First characterization of the vesicle components revealed that XPAPC is
internalized via the clathrin-mediated endocytosis pathway.

Unterseher, F. et al., EMBO J 23, 3259-60 (2004).
Wallingford J.B. et al., Nature 405(6782): 81-5 (2000).

Intercalation and delamination in the ectoderm of Xenopus laevis embryos:
morphometrical measurements and modeling.
 S. Kremnev. Laboratory of Developmental Biophysics, Department of Embryology, Moscow
State University. E-mail: s.kremnyov@googlemail.com

         One of the mostly wide spread morphogenetic processes are exemplified by
formation of so called delamination furrows, DF (straightening of cell-cell borders) and cell
intercalation, CI (insertion of cells between each other associated with a complete change of
cell-cell contacts). The both processes are initiated within undifferentiated embryonic tissue
which consists of tightly packed isodiametric cells. To a first approximation, such a tissue can
be considered as a hexagonal cell net. The aim of our work was to explore the mechanisms
leading to formation of DF and CI within such a homogeneous cell net exemplified by a
suprablastoporal area of midgastrula stage Xenopus laevis embryos.
  First, we observed that characteristic times for the formation of DF (no more than 20 min)
are in an order smaller than those required for CI (about 1 h). Then we elaborated a model
implying a feedback between the passive stretching and active contraction of actin-
containing cell edges [1]. According to the model, DF areas should contain a greater
percentage of apexes joining more than 3 edges. These expectations are confirmed by
morphometric measurements. Besides, we discuss the relations of our data to those
describing the formation of cell rosettes and CI in avian embryos and Drosophila blastoderm
[2, 3].

[1] Beloussov L. V., 2008, Mechanically based generative laws of morphogenesis, Phys.
Biol., 5:1-19
[2] Claire Bertet, Lawrence Sulak & Thomas Lecuit, 2003, Myosin-dependent junction
remodeling controls planar cell intercalation and axis elongation, Nature, 429:667-671
[3] Laura J. Wagstaff, Gemma Bellett, Mette M. Mogensen, and Andrea Münsterberg, 2008,
Multicellular rosette formation during cell ingression in the Avian Primitive Streak, Dev Dyn
237:91–96.


Specification of primary sensory neurons by β-catenin-mediated Wnt signaling.
Maria Krivega and Sergei Y. Sokol. Department of Developmental and Regenerative Biology,
Mount Sinai School of Medicine, New York, NY 10029, USA.

Wnt signaling regulates proliferation and differentiation of different cell types in vertebrate
embryos; however, its specific roles during neuronal differentiation have been elusive due to
the reutilization of this pathway at different developmental stages. Here we examined a role
for Wnt signaling in the specification of primary neurons in Xenopus embryos. Up- and
down-regulation of Wnt/β-catenin signaling in whole embryos led to corresponding changes
in the number of primary neurons, indicating the involvement of Wnt signaling in
neurogenesis. The observed effects required Wnt signaling after midblastula transition and
did not depend on early axis specification, mesoderm or neural induction. The analysis of
neuronal markers revealed a role of Wnt signaling in sensory, rather than motor or
interneuron differentiation. We further show that the paired-domain transcription factor Pax-
3, which is induced by Wnt signaling in the lateral neural plate, is required for Wnt-dependent
induction of Rohon-Beard sensory neurons, but is not sufficient to cause ectopic
neurogenesis on its own. We conclude that Wnt/β-catenin signaling functions through Pax-3
to regulate sensory neuron differentiation, independently of earlier developmental roles for
this signaling pathway.


A novel role of Syndecan-4 in neural induction by ERK- and PKC- dependent pathways
Sei Kuriyama and Roberto Mayor
Department of Anatomy and Developmental biology, University College London, Gower
Street, London WC1E 6BT, United Kingdom.

Syndecan-4 (Syn4) is a heparan sulfate proteolgycan that has been reported to control cell
migration and binds some growth factors, such as FGF. Here we describe a new role for
Syn4 during neural induction. We show that Syn4 is expressed in ectoderm competent to
respond to neural induction and when this competence is lost Syn4 become restricted to the
neural plate. Loss of function experiments using antisense morpholino oligonucleotides show
that Syn4 is required for the expression of neural markers in the neural plate and in
neuralized animal caps. Injection of Syn4 mRNA into ventral blastomeres or in animal caps is
sufficient to induce the expression of neural but not mesodermal markers. We found that two
parallel pathways activated by Syn4 are required for its neural inducer activity: First, an
FGF/ERK signaling pathway that can be inhibited by a dominant negative FGF receptor, by
the FGF inhibitor SU5402 and the MEK inhibitor U0126. Second, a PKC signaling pathway,
which is dependent on the intracellular domain of Syn4. We show that neural induction by
Syn4 throughout the PKC pathway requires the inhibition of PKC-delta and the activation of
PKC-alpha. Furthermore, we found Rac GTPase was negatively controlled by PKC-alpha
activation. Since PKC-delta/Rac/JNK pathway is essential to regulate the transcription of c-
jun, we examined if AP-1 complex is involved in PKC-alpha-dependent pathway. Finally, we
found PKC-alpha stabilized c-fos protein, and the activation of the inducible-c-fos after
gastrulation neuralized ventral ectoderm. Although some elements of PKC pathways have
been described during cell migration, we describe here that a similar pathways participate in
neural induction.

A functional screen for genes involved in Xenopus pronephros development
Jun-ichi Kyuno, Karine Massé and Elizabeth A. Jones
Department of Biological Sciences, Warwick University, Gibbet Hill Road, Coventry CV4 7AL,
UK

 Vertebrate kidney morphogenesis involves the progressive development of three kidney
forms; the pronephros, mesonephros and metanephros. In the amphibian Xenopus, the
pronephros is the functional larval kidney and consists of two identifiable components; the
glomus, the pronephric tubules, which can be divided into four separate segments, based on
marker gene expression. The simplicity of this organ, coupled with the fact that it displays the
same basic organisation and function as more complex mesonephros and metanephros,
makes this an attractive model to study the vertebrate kidney formation. In this study, we
have performed a functional screen specifically to identify genes involved in pronephros
development in Xenopus, a relatively late developmental event. The gain of function screens
are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length
plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone
that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage
cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric
marker expression examined by whole mount in situ hybridization or antibody staining. Most
of the positive clones had clear expression patterns in pronephros and predicted/established
functions highly likely to be involved in developmental processes. In order to carry out a more
detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly
specific expression patterns in the pronephric region. The over-expression of these five
selected clones indicated that they caused pronephric abnormalities with different temporal
and spatial effects. These results suggest that our strategy to identify novel genes involved in
pronephros development was highly successful, and that this strategy is effective for the
identification of novel genes involved in late developmental events.



An increase in intracellular Ca2+ is involved in pronephric tubule differentiation in the
amphibian Xenopus laevis.

Catherine Leclerc1, Sarah E. Webb2, Andrew L. Miller2 and Marc Moreau1.
1
  Centre de Biologie du Développement, UMR 5547 and GDR 2688, Université Paul Sabatier,
118 Route de Narbonne, F-31062 Toulouse, CEDEX 04, France.
2
  Department of Biology, The Hong Kong University of Science and Technology, Clear Water
Bay, Kowloon, Hong Kong, PRC.

        The pronephros is the first kidney to develop and is the functional embryonic kidney in
lower vertebrates. It has previously been shown that pronephric tubules can be induced to
form ex vivo in ectodermal tissue by treatment with activin A and retinoic acid. In this study,
we investigated the role of Ca2+ signaling in the formation of the pronephric tubules both in
intact Xenopus embryos and ex vivo. In the ex vivo system, retinoic acid but not activin A
stimulated the generation of Ca2+ transients during tubule formation. Furthermore, tubule
differentiation could be induced by agents that increase the concentration of intracellular Ca2+
in activin A-treated ectoderm. In addition, tubule formation was inhibited by loading the
ectodermal tissue with the Ca2+ chelator, BAPTA-AM prior to activin A/retinoic acid treatment.
In intact embryos, Ca2+ transients were also recorded during tubule formation, and photo-
activation of the caged Ca2+ chelator, diazo-2, localized to the pronephric domain, produced
embryos with a shortened and widened tubule phenotype. In addition, the location of the
Ca2+ transients observed, correlated with the expression pattern of the specific pronephric
tubule gene, XSMP-30. These data indicate that Ca2+ might be a necessary signal in the
process of tubulogenesis both ex vivo and in intact embryos.


FGF receptor-induced phosphorylation of ephrinB1 modulates planar cell polarity
signaling through Dishevelled

Hyun-Shik Lee, Kathleen Mood, Yon Ju Ji, Gopal Battu, and Ira O. Daar

Laboratory of Cell and Developmental Signaling, National Cancer Institute-Frederick,
Frederick, Maryland 21702, USA.

EphrinB1 signals through its cytoplasmic domain to promote cellular movements into the eye
field, whereas activation of the FGF receptor represses these movements and retinal fate.
Recent evidence indicates that in Xenopus embryos, ephrinB1 plays a role in retinal
progenitor cell movement into the eye field through an interaction with the scaffold protein
Dishevelled (Dsh). However, the mechanism by which the FGF receptor may regulate this
cell movement is unknown. Here we present evidence that FGF receptor-induced repression
of retinal fate is dependent upon phosphorylation of tyrosines 324 and 325 within the
intracellular domain of ephrinB1. We demonstrate that phosphorylation of these residues
disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is
dependent on the planar cell polarity (PCP) pathway. These results provide mechanistic
insight into how FGF signaling modulates ephrinB1 control of retinal progenitor movement
within the eye field.

A novel Xenopus pronephric tubules-expressing gene, XPteg is essential for the
pronephros development involving retinoic acid signaling.

Seung-Joon Lee, Sanghee Kim, Narina Bae, and Jin-Kwan Han*
Department of Life Science and Division of Molecular and Life Sciences, Pohang University
of Science and Technology, San31, Hyoja Dong, Pohang, Kyungbuk 790-784, South Korea
*
    Corresponding author

Retinoic acid (RA) signaling is important for early steps of nephrogenic cell fate specification.
Here we report a novel gene which is exclusively localized in developing pronephric tubules
and plays a role in pronephros development involving RA signaling in the Xenopus embryos.
We refer to this gene as XPteg, Xenopus Pronephric tubules-expressing gene. The
expression of XPteg was first detected in the presumptive pronephric mesoderm at early
gastrula, confined to the pronephric anlage at neurula, and found only in the proximal
segments of pronephric tubules during tubulogenesis. Antisense oligo nucleotide, MO-based
knockdown of XPteg caused disruption of pronephros formation and reduced expression of
pronephric markers. Moreover, XPteg has a competence of inducing pronephric mesoderm
in activin-treated in vitro culture. This potential of XPteg to induce pronephric mesoderm and
functions in the development of pronephros are regulated by RA signaling as a direct target
of transcriptional activation. Epistatic analysis of XPteg and modified RA receptors (RARs),
DN RARα and RARα-VP16 revealed that a predicted membrane protein, XPteg functions
downstream on RAR in the pronephric tubulogenesis. Taken together, XPteg involving RA
signaling is essential for pronephric mesodermal specification and pronephric tubule
development during Xenopus embryogenesis.



Endocytosis is required for apical constriction in Xenopus bottle cells
Jen-Yi Lee and Richard Harland
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA
94720-3200

Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation,
and organogenesis. The beginning of Xenopus laevis gastrulation is marked by the apical
constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a
crevice at the blastopore lip. Bottle cells simultaneously undergo apical constriction and
apicobasal elongation, transforming from cuboidal cells into elongated cells with tapered
necks. How do cells undergo such dramatic shape changes quickly? We hypothesized that
endocytosis of the apical membrane could regulate the efficiency of apical constriction. To
investigate this, we used a biotin-labeling technique to follow endocytic activity in Xenopus
embryos. Biotin-labeled endocytic vesicles were observed exclusively in bottle cells,
suggesting that bottle cells are the only cells actively undergoing extensive endocytosis
during the onset of gastrulation. Moreover, we disrupted endocytosis by expressing a
dominant negative (DN) form of Dynamin, a small GTPase required for the scission of the
endosome from the membrane. DN Dynamin-injected embryos exhibited shallower
blastopore grooves and bottle cells that were less apically constricted, without affecting cell
fate or subsequent morphogenetic events. To independently confirm that endocytosis is
required for apical constriction, we injected DN and constitutively active (CA) forms of Rab5,
a small GTPase required for transport of early endosomes. Injection of DN Rab5
recapitulated the DN Dynamin results, whereas CA Rab5 had no effect on either blastopore
depth or apical constriction. Transcytosis does not contribute to apicobasal elongation, since
cell length was not affected by the perturbation of endocytosis with DN Dynamin or DN Rab5.
In summary, our results show that endocytosis is crucial for apical constriction in bottle cell
morphogenesis. More generally, our findings increase our understanding of how membranes
are rapidly reorganized during cell shape changes in development and disease.


The reciprocal repression of dorsoventral specific genes are required for Xenopus
patterning

Ok-Joo Lee1, Hyun-Shik Lee2, Seung-Hwan Lee1, Jaeho Yoon1, Sung Chan Kim1, Jae-Bong
Park1, Jae-Yong Lee1 and Jaebong Kim1
1
    Department of Biochemistry, College of Medicine, Hallym University, Chunchon, Korea
2
 Laboratory of Protein Dynamics and Signaling,, National Cancer Institute-Frederick,
Frederick, USA


In the developing Xenopus embryos, dorsal-specific genes are expressed only dorsal and
organizer region, and repressed within ventral territory. On the other hand, ventral-specific
genes are repressed within dorsal and organizer region. Since the proper transcriptional
regulation of BMP-4-responsive molecules, Xvent homeobox genes and BMP-4 antagonizing
organizer molecules, eg. noggin, are critical for dorsoventral specification during early
embryogenesis, the reciprocal repression of BMP-4-responsive molecules and BMP-4
antagonists were proposed in this study. The hypothesis was investigated using noggin
promoter and PV.1 mRNA. As expected, the injection of PV.1 mRNA significantly
downregulated noggin transcription and its promoter activity, indicating that the negative
operator region by PV.1 was present within noggin promoter. In addition, negative
transcriptional regulation of PV.1A promoter was examined using a specific transcription
factor, goosecoid which is considered to be dorsal-specific transcription factors. PV.1A
promoter activity was downregulated even in the presence of BMP-4. Together, the results
suggest that dorsal- and ventral-specific genes reciprocally repress the transcription of
ventral- and dorsal-specific genes, respectively.

Keywords: Xenopus, patterning, PV.1, Goosecoid, Noggin



The role of NPHP2 (Inversin) in pronephros development

Soeren Lienkamp, Athina Ganner, Daniel Romaker, Julia Siraky, Stefan Spreitzer, Verena
Gieloff, Joachim Gloy, Gerd Walz

Renal Division, University Hospital Freiburg, Freiburg 79106, Germany


Nephronophthisis is the most common genetic cause of renal failure in children and young
adults. Changes in kidney morphology include tubular atrophy, basement membrane
thickening and cortico-medullary cysts. The disease can be associated with extra renal
manifestations including neural defects, blindness and situs inversus. At least 9 genes have
been found to cause nephronophthisis (NPHP1-9). In a previous study we identified NPHP2
(Inversin) as a regulator of Wnt-signalling and essential for convergent extension
movements. In this study we sought to determine the role of NPHP2 in early pronephros
development of Xenopus laevis. We found that knockdown of Xenopus NPHP2 caused
severe edema when targeted to the prospective pronephric tissue, suggesting defective
pronephros development. In situ hybridisation for the early pronephros marker pax-8
revealed that the early pronephric anlagen were not affected by NPHP2 knockdown.
However, immunostaining with pronephros specific antibodies at later stages displayed a
severe reduction in tubular staining. Microinjection and monitoring of the excretion of
fluorescent dyes in vivo revealed that the tubular lumen was patent and urinary excretion
occurred despite impaired coiling and elongation of tubules. Fz8 activates the non-canonical
Wnt-singalling pathway and has been shown to be essential for pronephros development.
Interestingly, NPHP2 partially rescued the pronephric defects caused by xFz8 knockdown,
indicating that NPHP2 acts downstream of xFz8 during renal development. Future studies
will determine how activation of non-canonical Wnt-signalling contributes to shaping the
pronephros. We aim to identify downstream effectors and will address how defects in other
NPHPs contribute to renal pathology and cystic kidney disease.




Mechanisms of cranial remodelling during metamorphosis
Bethany J. Slater1, Matthew D. Kwan1, Natalina Quarto1, Michael T. Longaker1 and Karen J.
Liu2
1
  Stanford University School of Medicine and 2Dept of Craniofacial Development, King’s
College London

         The tremendous diversity in vertebrate skull formation illustrates the range of forms
and functions generated by varying genetic programs. Understanding the molecular basis for
this variety may provide us with insights into mechanisms underlying human craniofacial
anomalies. The head structures of Xenopus undergo dramatic remodelling during
metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. To
investigate Xenopus calvarial morphology, we have examined late larval, metamorphosing,
and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-
computed tomography (mCT) as well as tissue staining, we have analyzed the development
of the frontoparietal bone and the fate of the surrounding cartilage. We observed that bone
formation initiates from lateral ossification centers, proceeding from posterior-to-anterior.
Histological analyses revealed midline abutting and posterior overlapping sutures. We then
set out to determine the mechanisms underlying the large-scale cranial remodelling during
metamorphosis. We examined proliferation, apoptosis, and proteinase activity during
remodelling of the skull roof. We found that tissue turnover during metamorphosis could be
accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-
1 and -13. Our studies demonstrate the feasibility of Xenopus as an alternative model system
for studying cranial development and tissue remodelling.


MiR-124a overexpression induces malformation of eye and targets Lhx2 in Xenopus
laevis
1
    Rong Qiu, 1Ying Liu, 2Jane Y. Wu and 1Rongqiao He
1
 The State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese
Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China
2
 Department of Neurology, Lurie Comprehensive Cancer Center, Center for Genetic
Medicine, Northwestern University Feinberg School of Medicine, 303 E. Superior, Chicago,
IL 60611, USA

It has been reported that miR-124a is abundant in the central neural system including eye
and is related to neurogenesis in different species. However, the role of miR-124a in eye
remains unclear. We show here, in Xenopus laevis, the expression of miR-124a begins along
the neural fold including the protruding eye anlagen at mid-neurula (stage 18) and continues
to adult stages at a high level in the entire central neural system including the eye, exhibiting
an early-low and later-high expression pattern. Microinjection of miR-124a precursor at 8-cell
stage leads to the malformation of the optic nerve and optic cup, indicating the importance of
keeping the miR-124a level low at the early embryonic stage. In addition, miR-124a
overexpression obviously downregulates the expression of Lhx2, Hairy2, Gli3, NeuroD1 and
Otx2 in/around eye anlagen, and Lhx2 could be directly targeted by miR-124a as shown by
luciferase assays. These results indicate that miR-124a is able to provide post-transcriptional
regulation on the genetic network directing eye morphogenesis during early Xenopus
development, and this mechanism of the early interaction might also persist in the role of
miR-124a in maturing and adult eye and brain.

Key words: MiR-124a, Eye, Lhx2, Xenopus laevis

Note This work was supported by the NSFC (No. 30771129, 30530280) and National Basic
Research Program of China (973 Program 2005CB522804).




Pou-V regulatory network in embryonic development.
Alessandra Livigni, Fella Hammachi, Alexei Sharov*, Gillian M. Morrison, Minoru Ko* and
Joshua M. Brickman.
MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, University of
Edinburgh; *National Institute on Aging, NIH, Baltimore.

The POU-V transcription factor Oct4 is a master regulator of self-renewal and pluripotency in
embryonic stem (ES) cells as well as an important regulator of lineage commitment in
embryonic development. We have shown that Oct4’s ability to regulate self-renewal in ES
cells is related to a conserved function in regulating embryonic differentiation in certain
vertebrates. In Xenopus, there are three POU-V proteins related to the mouse Oct4
(Xlpou25, Xlpou60 and Xlpou91). Interestingly, one of these proteins, Xlpou91, is able to
replace the murine Oct4 in regulating ES cell self-renewal. Here we explore the conserved
regulatory networks downstream of Oct4 and Xlpou91. While, Oct4 can function as an
activator or a repressor, we show that the activator function of POU-V factors plays the major
role in the suppression of lineage specification suggesting that the conserved regulatory
network downstream of these proteins is the key to how they regulate differentiation. Thus
the expression of activator fusion proteins, Oct4-VP16 or Xlpou91-VP16 inhibits embryonic
differentiation in Xenopus and supports ES cell self renewal in the absence of Oct4. Gene
expression profiling of the ES cells expressing Oct4-VP16 indicates that genes negatively
regulated by Oct4 are negatively regulated by Oct4-VP16, indicating there is little evidence
for a dominant role for these proteins as repressors. This microarray dataset also indicates a
number of interesting regulators that are Oct4-vp16 responsive and contain homologues in
Xenopus. To determine the subset of these regulators that are conserved in evolution, we
have extended our previous loss of function studies in Xenopus with new morpholino
combinations and use microarray analysis to investigate the role of POU-V factors in
controlling mechanisms of lineage specification. Through heuristic cluster analysis we
compare amphibian and murine ES cells datasets to define the evolutionary conserved POU-
V-dependent molecular network controlling germ layer specification.




Notch signaling during early Xenopus development

Diego Revinski, Alejandra Paganelli, Andrés Carrasco and Silvia López

Laboratorio de embriología molecular. Instituto de Biología Celular y Neurociencias. Facultad
de Medicina, UBA. Paraguay 2155 Piso 3 (1121) Ciudad Autónoma de Buenos Aires.
Argentina


We have found that activating Notch signaling from early cleavage stages results in
complementary changes in the expression of Sox2 and myoD or myf5 at neural plate stages.
This suggested that Notch may favor neural development at the expense of paraxial
mesoderm. The competence for Notch signaling to attain these complementary changes
finishes around stage 10. Knock-down of Notch signaling with a morpholino oligonucleotide
moves the sox2 expression domain further away from the blastopore at mid gastrula and
increases Xbra expression, consistent with the idea that Notch may be refining the limit
between neuroectoderm and mesoderm. Continuous treatment of embryos with DAPT, an
inhibitor of γ-secretase, increases myoD while inhibits sox2 expression in neurulae.
Moreover, treatment with DAPT from stage 1 to stage 8 is sufficient to promote this increase
of myoD, suggesting that Notch signaling is active in Xenopus embryos at earlier stages than
previously recognized and that it may be refining the limit between neuroectoderm and
paraxial mesoderm before gastrulation movements begin.

Understanding the control of gastrulation movements: Role of the Nodal signaling
pathway

Luxardi Guillaume, Leslie Marchal, Pierluigi Scerbo, Laurent Kodjabachian.
Institut de Biologie du Développement de Marseille-Luminy, CNRS Université de la
Méditerranée UMR 6216, Parc Scientifique de Luminy, Case 907 13288 Marseille Cedex 09,
France, e-mail : luxardi@ibdml.univ-mrs.fr

         Nodal-related ligands of the Transforming Growth Factor-beta (TGF-β) superfamily
play central roles in mesendoderm induction, organiser specification, and establishment of
left-right asymmetry. New roles for this pathway in the maintenance of embryonic stem cell
pluripotency, and in carcinogenesis have been shown more recently1.
         In this study, we investigate the possibility that Nodal signalling controls gastrulation
movements, independently of its role in mesendoderm induction. In order to temporally
control the activity of the pathway, we used SB431542 and SB505124, two reversible
selective chemical inhibitors of Alk4,5,7 receptors, as well as Nodal or its antagonist Lefty
protein injection in the blastocoele of blastula and gastrula embryos.
         Using the inhibitory reagents, we first established that the requirement for Nodal
signalling in mesendoderm fate establishment ends at late blastula stages. We found,
however, that active Nodal signalling is required from late blastula until mid-gastrula for
correct gastrulation movements, independently of mesendoderm fate maintenance. We
showed that Nodal signalling is autonomously required for chordamesoderm convergence
extension and prechordamesoderm migration, mediating cell-matrix and cell-cell adhesion.
Taking advantage of an Affymetrix DNA chip dataset that we generated to look for novel
Nodal target genes, we could show that the expression several known movement effector
genes depends on Nodal signalling during gastrulation. Moreover, Nodal recombinant protein
can activate the expression of several of these genes in the presence of cycloheximide,
suggesting that they are direct transcriptional targets of the pathway. Morpholino mediated
knockdown indicates that Xnr1 and Xnr2 together control this gastrulation specific program,
while they play little role in mesendoderm induction.
        Altogether, this work reveals that Nodal signals first regulate mesendoderm
development, and subsequently control gastrulation movements, where they may function at
least partly via the transcriptional control of critical effectors.


1.Nodal signaling: developmental roles and regulation. Michael M. Shen, Development 134,
1023-1034 (2007).




Title: Identifying targets of MyoD & Myf5 in myogenic stem cells

Authors: Maguire RJ, Isaacs HV, Pownall ME

Affiliation: Department of Biology, University of York, YORK, UK

Abstract:
The basic-helix-loop-helix transcription factors MyoD and Myf5 have an important and central
role in muscle development, which is highly conserved across many classes of animal.
Recent in vitro cell culture studies have shown a role for these transcription factors not only
during muscle differentiation, but also in proliferative myoblasts.
        In the frog species Xenopus laevis, an easily accessible in vivo population of
proliferative myoblasts is available for a relatively long period of time, during gastrulation.
This population of proliferative myoblasts also expresses both MyoD and Myf5 long before
the earliest marker of differentiating muscle, α-cardiac actin. In this poster, I present some
results from a microarray screen to identify the targets of these genes at this time point.

Zic genes act directly downstream of FGF signalling and BMP inhibition to promote a
                            pre-neural state in Xenopus

L. Marchal , G. Luxardi and L. Kodjabachian
I.B.D.M.L – Case 907 – 163 Avenue de Luminy – Marseille 13288 cedex 09 France
marchal@ibdm.univ-mrs.fr

Neural induction is the process that initiates nervous system development in vertebrates.
Two distinct models have been put forward to describe this phenomenon in molecular terms.
The default model states that ectoderm cells are fated to become neural in absence of
instruction, and do so when BMP signals are abolished. A more recent view implicates a
conserved role for FGF signalling that collaborates with BMP inhibition to allow neural fate
specification. Using the Xenopus embryo, we provide novel and definitive evidence in favor
of the second combinatorial model. We show that a dominant-negative R-Smad, Smad5-
somitabun, unlike the other BMP inhibitors used previously, can trigger conversion of
epidermis into neural tissue in vivo. It does so, however, only if FGF activity is also present,
suggesting that the two pathways control different effector genes required for neural
specification. In support of our combinatorial view, we report the isolation of the first two
direct neural targets of FGF signalling, zic3 and foxD5a, and of a direct neural target of BMP
inhibition, zic1. We showed that zic1 and zic3 are required together for definitive neural fate
acquisition, both in ectopic and endogenous situations. We propose that the combination of
FGF signalling and BMP inhibition regulates a pre-neural state marked by early zic gene
expression, that is a necessary first step for the emergence of stable neural progenitors.




The ectonucleotidase ENPPs and their roles during Xenopus development
Karine Massé, Jun-Ichi Kyuno, Surinder Bhamra, Nicholas Dale and Elizabeth A. Jones.
Department of Biological Sciences, Warwick University, Coventry CV47AL, UK.

The purines, ATP, ADP and adenosine, are important signalling molecules in the nervous
system but also in non neuronal tissues. ATP is sequentially degraded to adenosine by the
ectonucleotidase proteins. As ATP, ADP and adenosine activate distinct receptors, the
ectonucleotidases occupy a central role balancing the actions of these bioactive molecules.
Three gene families are prevalent to hydrolyse purines nucleotides, the E-NTPDase gene
family, the CD73 gene family and the ENPP (or PDNP) gene family. We have already
studied the E-NTPDase gene family in Xenopus embryos and discovered an unsuspected
role for the E-NTPDase2 ATPase and the purine pathway during eye development.
Here, we report the cloning of all 8 members of the E-NPP family, which encodes ecto-
phosphodiesterase/nucleotide phosphohydrolases, in Xenopus laevis and tropicalis. These
enzymes catalyse the hydrolysis of purine nucleotides but also the generation of bioactive
lipids. Our phylogenetic analysis shows this family is highly conserved between the frog
species but also during vertebrate evolution. In the adult frog, ENPP genes are broadly
expressed. During development, all ENPP genes are expressed and display distinct specific
expression pattern, suggesting potential different functions of these proteins during
embryogenesis of Xenopus laevis. Specially, several ENPP genes are highly expressed in
the embryonic kidney, the pronephros and their misexpression in Xenopus laevis affects the
formation of this organ. Furthermore, we are also characterising the distribution of the P2X
and P2Y purinoreceptors and the LPA and S1P bioactive lipids receptors in order to identify
which ones are expressed in the kidney and transduce the bioactive lipid/purine signalling
generated by the ENPP enzymes.




Ets and Kruppel-like factors cooperate to activate Flk-1 expression during Xenopus
vascular development.
Stryder M. Meadows, Matthew C. Salanga and Paul A. Krieg
Department of Molecular, Cellular and Development biology. The University of Arizona.

The receptor tyrosine kinase, Flk-1, is critical to the formation of blood vessels. Mice lacking
Flk-1 fail to develop the endothelial cell lineage. Identification of the factors that regulate Flk-
1 expression may lead to the molecular mechanisms responsible for the initial formation of
the primary vasculature. Using comparative genomics, we have identified sequences within
the previously reported Flk-1 intronic enhancer that are conserved between human and frog.
These sequences correspond to binding elements for Ets and Kruppel-like transcription
factors (KLF). Mutation of either sites in Xenopus transgenics results in dramatic reduction
of reporter gene expression. Over-expression of KLF2 or Erg, is sufficient to induce Flk-1
expression in avascular regions in the frog embryo, while inhibition of KLF2 function results
in dramatic reduction in expression of Flk-1 in vivo. Furthermore, KLF2 and Erg cooperate to
promote the transcription of Flk-1. These experiments are the first to indicate cooperation
between Ets and KLF transcription factors during vascular development.
Direct targets of the Zic1 transcription factor in early neural development
E. Jean Cornish, Sabah M. Hassan, and Christa Merzdorf
Department of Cell Biology and Neuroscience
Montana State University; Bozeman, Montana, 59715; USA

The Zic1 transcription factor plays multiple roles during early development, for example in
patterning the early neural plate, development of the neural crest, somite development, and
formation of the cerebellum. To increase our understanding of the molecular mechanisms
that underlie the activities of Zic1 in development, we have conducted a DNA microarray
screen in Xenopus laevis to identify downstream target genes of the Zic1 transcription factor.
The screen was designed to discover direct targets of Zic1. One of these direct targets is the
gene for an aquaporin that is most closely related to aquaporin 3. However, unlike aquaporin
3, it is expressed maternally and throughout embryonic development beyond Xenopus stage
24. In adult tissues, it is uniquely expressed in the colon (aquaporin 3 is expressed in the
lung and both aquaporins are expressed at low levels in kidney and skin). In neurula stages,
it is expressed very specifically in the anterior neural folds, extending more posteriorly as the
neural tube begins to close. Aquaporins are transmembrane proteins that form water
channels in the plasma membrane and have been shown to facilitate cell movement and cell
shape changes, which are both needed for neural tube closure. We are currently
investigating whether this aquaporin may play a role in neural tube closure and if so whether
it is linked to the neural tube defects caused by reduced activity of zic genes. Further, we
will discuss other themes emerging from the results of our screen with respect to anterior-
posterior patterning and neural crest development. We will discuss these and give a general
overview of the genes identified in our screen.

Characterization of the Sirtuin-type protein family during early development of
Xenopus laevis
Marlen Metzig, Susanne Schuster, Thomas Hollemann
Inst. Physiol. Chemistry, Martin-Luther-University Halle-Wittenberg

The founding member of the Sirtuin-family was identified in yeast as an uncommon NAD+-
dependent deacetylase, which is probably involved in transcriptional silencing (Guarente
1999). When overexpressed, ySir2 was found to increases live span in yeast, whereas the
deletion of Sir2 led to a reduction of life span (Blander and Guarente 2004, Longo and
Kennedy 2006). Similar effects have been observed under caloric restriction and the
application of resveratrol, treatments which both led to an activation of ySir2 and vertebrate
Sirtuins. In vertebrates, seven SIRTUINs have been identified so far. They modify the activity
of a huge set of target proteins. Thus enable the cell to give a coordinated stress answer,
increase production and utilization of energy and force DNA-repair, which together increases
DNA stability and mediate resistance to cell damaging mechanisms. Little is known about the
function of Sirtuins in the development of vertebrates. In this study we investigated the role of
the Sirtuin family in the early development of Xenopus laevis. We present the spatiotemporal
expression and subcellular localization of all 7 Xenopus Sirtuins. By loss- and gain-of-
function approaches we test for their impact on mesodermal (XBra) and neural (XSox3)
development. Proliferation and apoptosis as well as gene expression analysis of cell cycle
regulator genes contribute to these investigations. Potential interactions between different
sirtuin members will be determined by GST-pull-down-assay. With our findings we hope to
achieve a better understanding of sirtuin function in the development of vertebrates.


Functional Analysis of Xenopus Oct4-related Factors.

Laura Michel & Ralph Rupp
Dept. of Molecular Biology, Adolf-Butenandt-Institut, Schillerstrasse 44, D-80336 Munich
The POU transcription factor Oct4 is one of a few key regulators of cellular pluripotency in
mammals. In Xenopus, three Oct4-related genes have been identified, which are maternally
and zygotically expressed from fertilization to neurulation. As shown by Morrison & Brickman
(Edingburgh), they can partially substitute for Oct4 in mouse ES cells. Furthermore, recent
work by the labs of Walter Knöchel (Ulm) and Dale Frank (Haifa) implicated these proteins in
multiple signaling pathways during germ layer formation, providing a conceptual framework
for further analysis of their functions during frog development.

Assuming that the three potential Oct4-homologs are part of a protein network similar to,
although not necessarily identical with, the mammalian Oct4/Sox2/Nanog triad, we have
generated a number of gain-of-function variants for overexpression studies. In the course of
this work, we have found that these proteins accumulate to very different levels in the
embryo, making it necessary to investigate the Oct4-homologs one by one. Here we will
present our results from injections of Xenopus Oct60-enR and –VP16 variants, which cause
with high penetrance the appearance of neomorphic tissue bulges at distinct positions along
the anteroposterior axes during neurulation. These structures are to some extend transient
and partially disappear before hatching stage, possibly correlated with the turnover of the
injected transcripts. Furthermore we have strong evidence that Xenopus Oct-60 has a
function in germ layer formation, especially in mesoderm and neuroectoderm, and influences
differentiation in Xenopus embryos as well as in animal caps.



Regulation for neural tube morphogenesis by Nectin, an Ig-like adhesion molecule
H. Morita1,2, C. Terasaka1, T. S. Yamamoto1, N. Ueno1,2
1 National Institute for Basic Biology, Okazaki, Aichi, Japan
2 The Graduate University for Advanced Studies, Hayama, Kanagawa, Japan

To better understand the developmental morphogenesis in vertebrates, we have investigated
roles of an adhesion molecule Nectin that is expressed in the early embryonic stages of
Xenopus laevis. We found that one type of Nectin, Nectin-2, belonging to the
immunoglobulin-like cell adhesion molecule, is expressed in neurula embryos, particularly in
the neural tube. Nectins in mammals are thought to organize the formation of epithelial cell
array and synaptic connection in the cultured cell system, but its role in developmental
processes is poorly documented. Therefore, in this study, we investigated in vivo role of
Nectins by depleting it with their specific morpholino antisense oligonucleotide. The effective
knockdown of Nectin caused neural tube defect manifested by the incomplete neural fold
formation and less accumulation of F-actin at the apical surface. We also found that these
are due to an aberrant apical constriction, a cell-shape change that is required for the neural
tube folding. Conversely, overexpression of Nectin-2 in Xenopus embryo induced ectopic
apical constriction presumably due to increased actin accumulation on the apical side. Given
that Nectin is anchored to actin filament through Afadin, a Nectin- and actin-binding protein, it
might coordinate a bridge between extracellular cell-cell interaction and internal cytoskeletal
dynamics. However, further experiments have revealed that intracellular Afadin binding is not
required but the extracellular domain is necessary for the constriction ability of Nectin-2.
Furthermore, we found Nectin-2 physically interacts with N-cadherin, which is associated
with F-actin in the intracellular space, synergistically enhancing apical constriction. Our
findings may provide a new insight into vertebrate neural tube morphogenesis and the
mechanism of apical constriction with spatiotemporally upregulated Nectin-2 expression in
neural tube and regulation of apical constriction by the transmembrane cell adhesion
molecules.
A novel synaptotagmin-like membrane protein acts as an endocytic adapter that is
required for internalization of the activated FGF receptor, for prolonged ERK
activation and for mesoderm induction in Xenopus.
Steve Jean, Michel Tremblay, Joëlle Baril and Tom Moss
Cancer Research Centre and Department of Medical Biology, Laval University, CHUQ Hôtel-
Dieu de Québec, Pavillon St Patrick, 9 rue McMahon, Québec, G1R 3S3, Canada.


Signalling via the Fibroblast Growth Factor Receptor FGFR1 has been implicated in many
aspects of development and differentiation as well as in angiogenesis, metastasis and
cancer. FGF signalling is particularly important for cell movements during gastrulation, for
mesoderm induction and the differentiation of somatic muscle. Endocytosis is an essential
step in signal transduction by several plasma membrane receptors. However, little is known
of how the FGF receptor is endocytosed nor of the role this process plays in signalling. Here
we show that prolonged activation of the ERK pathway is essential for Xbra induction in
Xenopus and that inhibiting endocytosis of activated FGFR1 leads to a rapid downregulation
of ERK activity and the loss of Xbra expression. We further show that E-Syt2, a member of a
small family of extended synaptotagmin-like proteins, is essential for the endocytosis of
activated FGFR1. Synaptotagmins are membrane proteins required at synaptic terminals for
rapid exo- and endocytosis of neurotransmitters. However, their function in other tissues or
other signalling pathways has not been observed to date. We show that E-Syt2 is required
for functional ERK signalling and for mesoderm induction in Xenopus. E-Syt2 is shown to
interact selectively with FGFR1 and with Adaptin-2 in an FGF-dependent manner and to
determine FGFR1 internalization via Clathrin-dependent endocytosis. As such E-Syt2
appears to act as a bridging protein and to catalyze the assembly of clathrin coated pits
around the activated FGF receptor. The data brings us a significant step closer to
understanding both the mechanism and the role of endocytosis in FGF signalling and
presents the first indication that members of the larger synaptotagmin family function as
endocytic adapters for growth factor receptors.

The Role of Hsl7/PRMT5 in Xenopus Neuronal Development
Adriana Mejia, Susan C. Howard, and Paul R. Mueller
Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249

Hsl7/PRMT5 is part of the large family of protein arginine methyltransferases that is
conserved from yeast to vertebrates. Hsl7 was originally identified in budding yeast for its
synthetic lethality with deletions of histone termini. However, Hsl7/PRMT5 is now known to
be associated with a myriad of cellular events including cell cycle control, stress response,
and transcriptional regulation. Recent studies in Xenopus cell-free egg extracts have
suggested Hsl7/PRMT5 promotes cell cycle advancement by promoting the destruction of
specific Wee1-like kinases. However, the exact function of Hsl7/PRMT5 remains a mystery
and our studies suggest that this function may vary later in development. By in situ and
qPCR analysis we find that Hsl7/PRMT5 has a dynamic expression pattern. During the
cleavage stages, Hsl7/PRMT5 is abundantly expressed throughout the embryo. However,
during gastrulation Hsl7/PRMT5 expression is drastically reduced. Hsl7/PRMT5 expression
reappears near the end of neurulation, but is limited developing neuronal precursor tissues.
Finally by the tailbud stage, Hsl7/PRMT5 is expressed strongly in the eye, brachial arches,
and portions of the brain. While overexpression of recombinant Xenopus Hsl7/PRMT5 has
failed to show any overt phenotype, morpholino-mediated loss of Hsl7/PRMT5 results in
various neuronal defects from improper neural tube closure to eye deficiencies. Together,
these studies provide a foundation for future experiments that will uncover the function of
Hsl7 during early embryogenesis.


Expression analysis of the Xenopus specific Ets-like gene kate
Frank Müller and Thomas Hollemann

kate (known as transcription factor Ets like) was found in an in situ database screen (NIBB).
It represents a gene with a highly conserved C-terminal Ets-domain with strong homology
(78% to xl-Ets-1 on amino acid level) to the Ets-domain of xl-Ets-1 and -2. In contrast, a
BLAST search with the N-terminal domain of kate only found a x. tropicalis homolog, and a
SMART search showed no known domains, indicating that kate is a Xenopus-specific Ets-
like gene. Ets transcription factors are involved in a multitude of developmental processes,
ranging from neuronal development, eye cell fate to hematopoietic development. Ets-factors
can thereby act as transcriptional regulators. Depending on their interaction partners, they
act as activators or repressors (or both). We found kate expression in blood and endothelial
progenitor cells, in a pattern largely resembling the pattern of the endothelial gene Xfli1
(Walmsley et al., 2002). At NF stage 23 kate is expressed in the ventral blood islands. This
expression is only transient and cannot be seen from NF stage 25 onwards. From NF stage
23 on kate expression can be seen at the dorsal lateral plate, beginning in the region of the
pronephric sinus. RT-PCR analysis showed weak maternal expression that increases at late
neurula stages (NF st 20) and is maintained until tadpole stages (NF st 42). Since the Ets-
domain can confer the DNA-binding capacity of Ets-family transcription factors, kate might be
a transcriptional regulator. To analyse the function of kate in vivo we will perform Gain-of-
function and Loss-of-function experiments in Xenopus embryos and analyse the effect of
kate on blood cell and endothelial development.


             Two distinct roles for Xenopus Frizzled 7 during heart development
                    Jo Mulvaney, Muhammad Abu el Magd and Grant Wheeler
         School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ. UK
Wnt signaling is required during cardiogenesis for specification of heart tissue and the
morphogenetic movements necessary to form a looped, multi-chambered heart. Canonical
Wnt signaling is required pre-gastrula in zebrafish to specify cardiac-mesoderm whilst non-
canonical signaling is required for cardiomyocyte differentiation. In Xenopus, Wnt 11 has
been shown to mediate the non-canonical signaling required for differentiation. Morpholino
mediated knock down of Wnt 11 gives a heartless phenotype. Wnt 11R, which is expressed
later, is also thought to signal through the non-canonical pathway to promote heart
morphogenesis. Morpholino mediated knock down of Wnt 11R gives a cardia bifida
phenotype. Xenopus Frizzled-7 (Xfz7) is expressed during gastrulation in the mesodermal
cells fated to become heart. Expression is maintained in the primary heart field as the
dorsolateral mesoderm migrates to the ventral midline. As the heart continues to form,
expression continues in the myocardium, and later becomes restricted to the pericardium.
Morpholino mediated knock down of Xfz7 gives a heartless phenotype similar to that of a
Wnt 11 morpholino. It has been speculated that Wnt11 signals through Xfz7 in this context
but we show that they are acting in distinct pathways during heart field specification.
Surprisingly, expression of dominant negative Xfz7 (Xfz7 CRD) results in a cardia bifida
phenotype similar to Wnt 11R morpholino. We show that Xfz7 mediates Wnt 11R signaling in
contrast to Wnt 11 signaling. We propose that Xfz7 potentially interacts with a number of
Wnts and is necessary for at least two different events during heart development. Pre
gastrula Xfz7 mediates specification of the mesodermal cells that will form the heart, possibly
by interacting with an unknown Wnt to signal through the canonical pathway. Later, post
gastrulation, it signals through Wnt 11R and the non-canonical pathway to control fusion and
morphogenesis.


 p21-activated kinase controls cell polarity and directional mesendoderm migration in
                                   the Xenopus gastrula
Martina Nagel1, Olivia Luu1, Bojan Macanovic1, Tom Moss2 and Rudolf Winklbauer1,*
1
 Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto,
Ontario M5S 3G5, Canada. 2Cancer Research Centre and Department of Medical Biology,
Laval University, CHUQ-Hotel-Dieu de Quebec, 9 rue McMahon, Quebec, Quebec G1R 2J6,
Canada
The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility.
Using kinase-dead or constitutively active mutants of xPak1, we show that during Xenopus
gastrulation, the kinase activity of Pak is required for the establishment and maintenance of
cell polarity in the migrating mesendoderm. Pak induces cell polarization upstream of Cdc42.
Overactivation of Pak compromises maintenance of cell polarity, probably by increasing Rac
activity and inhibiting RhoA function. Inhibition of cell polarity does not affect the migration of
single mesendoderm cells. However, Pak inhibition interferes with the guidance of
mesendoderm migration by directional cues residing in the extracellular matrix of the
blastocoel roof, and with mesendoderm translocation in the embryo.


A role for the Notch signalling pathway in development of the Xenopus laevis
embryonic kidney, the pronephros.
Richard W Naylor and Elizabeth A Jones

          The Notch signalling pathway is one of six major signal transduction pathways in the
cell. It is involved in many developmental processes due to its ability to direct cell fates, cell
death and cell proliferation through three main modes of action; lateral inhibition, boundary
formation and asymmetrical inheritance. Through over-expression and morpholino
oligonucleotide approaches we propose the Notch signalling pathway establishes a border in
the dorso-anterior region of the X.laevis pronephros at early tail bud stages of development.
This border corresponds to the region of the lateral pronephric mesoderm that distinguishes
the nephrostomes from the proximal tubules. We show for the first time that the Notch
signalling modulators Lunatic Fringe and Radical Fringe are both expressed in this region of
the pronephros, and disrupting their expression affects pronephros development. Finally we
show that the Notch signalling pathway induces the Wnt signalling pathway to enable
differential gene expression and generate cellular fields within the anlagen that will
differentiate into three specific functional regions of the pronephros; the glomus,
nephrostomes and proximal tubules.

A RNA-binding protein encoded by a calcium-induced gene is involved in eye
development in Xenopus laevis embryo.

Isabelle NEANT, Nina DEISIG, Pierluigi SCERBO, Marc MOREAU
Centre de Biologie du Développement, CNRS-UMR5547, and GDR 2688, Université P.
Sabatier, 118 route de Narbonne, F-31062 Toulouse cedex, France

In Xenopus embryo an increase in intracellular concentration of calcium is necessary and
sufficient to drive dorsal ectodermal cells into neural fate at the onset of gastrulation (Leclerc
et al, 2000; 2006). We focused on the downstream genes responsible for the engagement of
the ectoderm into neural fate, and isolated such early calcium-dependent genes by a
subtractive library, constructed between competent ectoderms oriented into epidermal fate
(untreated, control ectoderms) and ectoderms that are oriented into neural fate by a caffeine-
induced [Ca2+]i burst (Batut et al, 2003; 2005). More than 30 calcium triggered-genes were
isolated. All of these genes exhibit expression patterns specifically restricted to the neural
territories during early development. Among these genes we have selected one, which
encode for a RNA binding-protein. The messenger is strongly expressed in anterior neural
structures during early development. Overexpression of the messenger leads to an alteration
of eye organogenesis and in the more severe phenotypes, to eyeless embryos.
Batut J, Néant I, Leclerc C, Moreau M. (2003). xMLP is an early response calcium target
gene in neural determination in Xenopus laevis. J Soc Biol.;197:283-289.
Batut J, Vandel L, Leclerc C, Daguzan C, Moreau M, Néant I. (2005). The calcium-induced
Arginine N-Methyltransferase xPRMT1b triggers neural determination in Xenopus laevis
embryos. Proc. Nat. Acad. Sci.(USA); 102:15128-15133.
Leclerc C, Webb SE, Daguzan C, Moreau M, Miller AL. (2000). Imaging patterns of calcium
transients during neural induction in Xenopus laevis embryos. J Cell Sci;113:3519-3529.
Leclerc C, Néant I, Webb SE, Miller AL, Moreau M. (2006). Calcium transients and calcium
signalling during early neurogenesis in the amphibian embryo Xenopus laevis. Biochimica et
Biophysica Acta; 1763:1184-1191.



Generation of Transgenic Xenopus laevis Isogenetic Clones with
Modulated Expression of MHC Class I
Hristina Nedelkovska1, William J. Bowers2 and Jacques Robert1
1
  Department of Microbiology & Immunology, 2Neurology, Center for Neural Development and
Disease, University of Rochester Medical Center, Rochester, NY 14642, USA

Xenopus laevis has been an important and unique animal model for immunological studies
for many years. One of the hallmarks of this model relates to the availability of inbred strains
as well as different MHC-defined isogenetic clones. Two of the clones that we have
available, LG-6 and LG-15, are instrumental to study tumor immunity and skin graft
rejection/tolerance. To further advance our model, we began to generate transgenic clones.
For this purpose we used the three recently established transgenesis techniques (the ΦC31
integrase, the Sleeping Beauty transposase and the meganuclease) since these clones are
generated by gynogenesis where the oocyte is only activated by UV-irradiated sperm, which
does not contribute to the genetic make up of the offspring. We tested all 3 techniques and
were able to generate GFP-expressing clones: both LG-6 and LG-15. Our goal, however, is
to upregulate or knockdown certain immunologically relevant genes such as MHC class I and
beta 2 microglobulin (β2-m). We, therefore, generated plasmids for all 3 platforms that either
contain our genes of interest or an shRNA transgene targeting the X. laevis MHC class I or
β2-m. Our preliminary results suggest that expression of anti-β2-m shRNA is not deleterious
in vivo and significantly knocks down β2-m in a number of transgenic clones. We are
currently testing whether the integrated transgenes will be passed through the germ line.
This will be a great advantage because each embryo will carry the transgene since they are
generated by gynogenesis, which will eliminate the need for further screenings and will
greatly increase the number of transgenic progeny.

Research Support: T32-AI-07285, R01-CA-108982-02, R24-AI-059830-01, and R01- AG-
023593 from the NIH


Testing cellular pluripotency by single cell transplantation

Dario Nicetto, Ralph Rupp
Department of Molecular Biology, Adolf-Butenandt-Institut, Schillerstrasse 44, D-80336
München

One of the most interesting aspects of developmental biology is the understanding of the
mechanism by which cells derived form a fertilized egg become restricted to a particular cell
type. By single cell transplantation experiments, Snape and colleagues demonstrated that
early animal pole blastula cells are pluripotent and become committed to form ectoderm
during mid and late blastula stages (Snape A., et al., 1987), describing the timing and
sequence of their determination. Here we have modified this assay: single Alexa fluor
dextran labelled animal pole blastomere is transplanted into a late blastula host embryos
blastocoel (stage NF 9). At stage 41 normal developed embryos are first examined at
fluorescence steromicroscope, and than embedded in low melting agarose and analyzed at
confocal microscope. This assay allows us to analyze whole-mount transplanted embryos
and in theory it can be used to test the state of commitment of cells taken from embryos at
different stages. Homochronic and heterochronic transplantations can be performed: the
former refers to the transplantation of cells in a particular stage in host embryos of the same
stage; the latter concerns the injection of cells taken from donor embryos younger or older
than host embryos. Although the transplanted animal pole cells contribute predominantly to
ectoderm during normal development, upon transplantation the same blastomeres give rise
to progeny detectable in all the three germ layers. When younger donor cells (NF 7-8) are
transplanted in host embryos, most of the blastomeres show pluripotent behaviour giving rise
to cells derivatives in ectoderm, mesoderm and endoderm, while none of them is committed
to form only ectoderm. Upon homochronic transplantation, late blastula (NF 9) donor cells
already show less pluripotency compared to mid-blastula cells; moreover some of the
transplanted blastomeres give rise to daughter cells restricted to ectoderm only, suggesting
that at this stage donor cells start being committed to their “default” fate. Donor cells from
stage NF 10.5 were transplanted in a series of heterochronic transplantation experiments
into the blastocoel of embryos at stage NF 9; unfortunately the problems of injecting such
small cells, and the less proliferative capacity of the same cells at this stage, make the
behaviour of the cells difficult to analyze. In general transplantation experiments provide a
useful tool to investigate cell behaviour at single cell level, representing a helpful assay to
study the role of key players involved in lineage commitment processes.


Functional analysis of NumbL during Xenopus neurogenesis.
Frank Nieber, Tomas Pieler and Kristine Henningfeld. Department of Developmental
Biochemistry, University Goettingen, Justus-von-Liebig Weg 11, 37077, Goettingen,
Germany.

In Xenopus, the first neurons, termed primary neurons, are born within the induced
neuroectoderm in three longitudinal domains on both sides of the midline shortly after
gastrulation. The number of neurons is restricted by lateral inhibition, mediated by the Notch
signalling pathway. In other model systems, members of the Numb protein family have been
described to inhibit the Notch signalling pathway by promoting either endocytosis of the
Notch receptor or proteasomal degradation of NICD, the intracellular effector of Notch
signalling.
To determine if the Numb proteins play a role during primary neurogenesis, we isolated
Xenopus NumbLike (NumbL). NumbL transcripts were first detected by whole mount in situ
hybridization at gastrula stages in the midline. At the open neural plate stage, NumbL
expression persisted in the midline and was additionally present in the territories of primary
neurogenesis. At later stages, NumbL was found in the brain, the eye, the neural crest and
the spinal cord. In whole embryos, NumbL was positively regulated by X-ngnr-1 and inhibited
by the Notch pathway. A knockdown of NumbL using antisense MO inhibited neuronal
differentiation demonstrating NumbL is essential for neurogenesis. The knockdown effect
could be rescued by coinjection of Xenopus NumbL and mouse NumbL RNA. Of the four
splice variants of mNumb, only mNumb4 rescued the NumbL MO phenotype. Interestingly,
the inhibition of neurogenesis in the absence of NumbL appears not to be the consequence
of an increase in the inhibitory Notch signalling pathway. In summary, the expression,
regulation and knockdown experiments demonstrate that NumbL plays a critical role during
the process of Xenopus primary neurogenesis.


Characterization of formation of single epithelial layer in Cynops pyrrhogaster early
embryo
Hiromasa Ninomiya, Rudolf Winklbauer
(Department of Cell & Systems biology, University of Toronto)

Selecting to be an epithelial or a mesenchymal cell type is involved in many developmental
processes. During germ layer formation, vertebrate embryos form ectoderm that consists of a
layer of epithelial cells with or without a mesenchymal cell layer below, but the significance of
this difference is not much explored. In amphibians, Xenopus laevis animal side of cells
consists of epithelial and mesenchymal cell layers and most of mesodermal and neural
tissues are formed from latter. On the other hand, a single epithelial cell layer is formed in
Cynops pyrrhogaster animal cells, and most of mesodermal and neural tissues are formed
through epithelial-mesenchymal transition. We have studied the formation of the ectoderm
layer in this species and compared it with that of Xenopus. We found two mechanisms to
keep the ectoderm a single layer. First, observation of cell divisional planes showed that
mitosis is preferentially oriented in the surface plane throughout early development. Second,
cell lineage analysis indicated that inner cells formed from animal cells are integrated into the
epithelial layer by radial intercalation. Immunostaining of aPKC and expressing Lgl-GFP
proteins suggested that there are peculiar cell surface domains in ectoderm. To clarify the
significance of a single layered animal cells for the development of Cynops, we induced the
formation of an inner layer of cells artificially by coexpression of aPKC and Lgl in the
epithelial layer. Interestingly, this thickening in head ectoderm resulted in heavier
morphogenetic defects in Cynops, while the thickening in dorsal marginal zone resulted
heavier axis elongation defects in Xenopus, suggesting different regional sensitivity for
thickening of the layer. These results suggest that the difference in animal cell layer structure
is accompanied by several changes in cell behavior and developmental mechanisms.

A European Xenopus Resource Centre.
 Anna Noble, Liz Jones* and Matt Guille. EXRC, School of Biological Sciences, University of
Portsmouth, UK. *University of Warwick, UK.
The centre has now been established for a year and has supplied large numbers of wild-type
animals and a few transgenic ones. We have received transgenic lines and some mutants
from UK and US labs and are in the process of expanding these ready for supply. More
transgenic and mutant lines are welcome. Transgenesis, gynogenesis and sperm freezing
are all established at the centre and the effect of different vector designs is being assessed.
Recently funding has been obtained to allow EXRC to curate and distribute Xenopus
plasmids and antibodies. This will also allow us to distribute the X. laevis characterised ESTs
and X. tropicalis fosmid libraries that have arrived from the US. The EXRC website and
ordering system will be accessed via Xenbase and this should be live prior to the meeting.
Key input from Xenopus researchers is needed regarding the techniques and the specific
lines that they want the centre to develop.
Much of this year has been taken up dealing with the possible restriction on the movement
and sale of Xenopus due to their ability to carry the chytrid fungus “Bd” that is a cause of
mass die-offs and even extinction of some amphibian species. The UK Xenopus colonies are
being tested for Bd and the overall results of this testing will be shown.

Genomic Synteny of Xenopus Immune-related Genes
Yuko Ohta1, Tereza Tlapakova2, Vladimir Krylov2, and Martin F. Flajnik1
1
  University of Maryland, Baltimore, Department of Microbiology and Immunology, USA
2
  Charles University in Prague Faculty of Science, Dept of Cell Biology, Prague, Czech
Republic

Evolutionarily, Xenopus species shared a common ancestor with humans ~350 million years
ago, and it is one of the high connectivity animals linking mammals to lower vertebrate taxa.
Through an in silico approach, we have uncovered many new genes that have important
roles in the Xenopus immune system and have analyzed their syntenic relationships relative
to other vertebrates. We found that, in contrast to teleost fish, the genomic synteny is
remarkably similar between the human and Xenopus (e.g. major histocompatibility complex
(MHC)s), yet in some cases apparent ancestral syntenies can be still found only in the
Xenopus genome. Thus, using a comparative approach, we will eventually comprehend the
natural history of the immune system. We predict the evolutionarily conserved genes might
have vital roles in fundamental immune function, whereas novel genes found only in
particular species, especially those within ancient linkage groups, could impart new insights
into the immune system. Furthermore, examining the novel genes in non-mammalian
models such as Xenopus might reveal the functions of their human homologues having
unknown or poorly described functions. We are particularly interested in the novel genes
that contain distinctive domains belonging to the variable (V) and the constant (C1)-type of
the immunoglobulin superfamily (IgSF), one of which resemble to B7 family costimulatory
molecules. In order to understand the phylogenetic relationship and the evolutionary history
of B7 gene family, we searched databases for the B7 orthologues and homologues from
different vertebrate classes, examined their syntenic relationships, and compared among
them. Our analysis revealed that the synteny is well conserved for some B7 genes,
suggesting these genes are phylogenetically older than genes with no syntenic conservation.
Furthermore, we found that the association of the B7 homologues to the Xenopus MHC,
suggesting the linkage of B7 to the MHC may be primordial.



TRIQK, a novel family of small proteins localized to the endoplasmic reticulum
membrane, is conserved across vertebrates.
Yasuko Onuma1, 2, Akira Watanabe3, Hiroyuki Aburatani3, Makoto Asashima4, 5,6 and Malcolm
Whitman1
1 Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood
Avenue, Boston, MA 02115, USA
2 Network for Life Science Education, The University of Tokyo, Tokyo, Japan
3 Genome Science Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo, Japan
4 Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The
University of Tokyo, Tokyo, Japan
5 ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), Tokyo,
Japan
6 National Institute of Advanced Industrial Sciences and Technology (AIST), Tsukuba,
Ibaraki, Japan

Here we report a novel small protein that is highly conserved across vertebrates. The protein,
which we have named TRIQK, has no homology to any previously reported proteins or
functional domains, but all vertebrate homologs of this protein share a characteristic triple
repeat of the sequence QXXK/R, as well as a hydrophobic C-terminal region. The Xenopus
triqk gene (xTriqk) was isolated in an expression screen on the basis of its ability to cause
dramatic changes in cell size and nuclear size and morphology in developing embryos. The
Xenopus and mouse triqk genes are broadly expressed throughout embryogenesis, and
mtriqk is also generally expressed in mouse adult tissues. TRIQK proteins are localized to
the endoplasmic reticulum membrane. Depletion of endogenous xTRIQK protein in Xenopus
embryos causes no detectable morphological or functional changes in tadpoles.

          Functional analysis of the hyaluronan-hyaluronan receptor system during
             neural crest cell migration and craniofacial morphogenesis

                      Paola Casini1, Roberto Perris2, Irma Nardi1, Michela Ori1
       1
        Unit of Cellular and Developmental Biology, Department of Biology, University of
       Pisa, Italy; 2Department of Genetics, Microbiology and Anthropology, University of
       Parma, Italy

         The cell, together with its extracellular matrix (ECM), represents a
     morphogenetic unit that is dynamically and coordinately modulated during
     embryonic development. Hyaluronan (HA) is a crucial glycosaminoglycan of
     vertebrate embryonic ECM and the control of its synthesis might influence cell
     behavior, both by assembling the interstitial matrices and by the activation of
     signal transducing receptors such as CD44.
          Recently we demonstrated a critical role of XHas2 and XCD44 during muscle
     formation and precursor muscle cell migration (1, 2). We are now focusing on
     cranial neural crest cells (NCC) development, knocking down the XHas1, XHas2
     and XCD44 gene functions. We revealed that the hyaluronan synthases and the
     hyaluronan receptor present a dynamic expression pattern during cranial NCC
     development suggesting multiple roles in the various steps of cranial NCC
     migration and differentiation. We showed that hyaluronan, synthesized by XHas1
     and XHas2, is necessary to a proper visceral skeleton development. In particular,
     hyaluronan presence around migrating cranial NCC, is necessary to keep an
     active migratory behavior, while its presence in the pericellular matrix surrounding
     pre-chondrogenic cranial NCC in the branchial pouches, is crucial for their
     survival before their terminal differentiation. XCD44 gene loss of function
     experiments also suggested that hyaluronan role, in the cranial NCC migration,
     could be mediated by the activation of XCD44 receptor. On the other hand
     XCD44 seems not to be required for skeletogenic precursors survival or terminal
     differentiation. In order to investigate possible action mechanisms underlying
     hyaluronan function, we are now exploring the possible functional interactions of
     hyaluronan with alternative receptors, such as RHAMM, and hyaluronan binding
     proteins such as the proteoglycan versican (3). These data, new and
     unpublished, demonstrated an unsuspected critical role of hyaluronan in the
     visceral skeleton morphogenesis and in particular in NCC migration and survival.

     1) Nardini M.,Ori M., Vigetti D., Gornati R., Nardi I., Perris R. (2004).
     Gene Expression Pattern 4(3): 303-308
     2) Ori M., Nardini M., Casini P., Perris R., Nardi I. (2006).
     Development 133(4): 631-640
     3) Casini P., Ori M., Avenoso A., D’Ascola A., Traina P., Mattina W., Perris R.,
     Campo G.M., Calatroni A., Nardi I., Campo S. (2008).
     Int. J. Dev. Biol. In press




Identifying Regulatory Elements of the Transcription Factor Tbx5 that Function During
Heart Development
Erika Paden1, 2, Benoit Bruneau4, 5, and Frank Conlon1, 2, 3
1
  Carolina Cardiovascular Biology Center 2UNC Department of Biology 3UNC Department of
Genetics 4Gladstone Institute of Cardiovascular Disease 5Department of Pediatrics UCSF

TBX5, a T-box transcription factor frequently mutated in patients with Holt-Oram syndrome,
plays critical roles during vertebrate heart development. In Xenopus depletion of TBX5
results in cardiac defects with aberrant sarcomere formation, perturbations in the timing of
cardiac differentiation, and a decrease in the number of cardiac cells. The decrease in
cardiac cell number in TBX5-depleted embryos is the result of cell cycle arrest at the G1/S
transition which eventually leads to programmed cell death. These severe defects are due to
the multiple roles for Tbx5 during cardiogenesis. Although Tbx5 is one of the earliest markers
of the cardiac field and its temporal and spatial pattern has been examined during
development, the mechanisms which control this process remain unknown. To begin to
address this question, we have identified two alternative transcriptional start sites that are
utilized during Xenopus cardiogenesis. We have examined the ability of regions surrounding
these start sites to induce expression of EGFP in the developing Xenopus heart. The timing
of expression from the transcriptional start sites as well as colocalization with TBX5 protein is
being examined. After establishing the regulatory elements critical for the expression of Tbx5
in Xenopus tissues, we examined the evolutionary conservation of the elements by
determining their ability to function during mouse cardiogenesis. We are utilizing these
potential regulatory elements to determine critical functional elements and the pathways
which lead to initiation of Tbx5 expression.

Sprouty3 in Xenopus development
Niki Panagiotaki, Karel Dorey, Nancy Papalopulu and Enrique Amaya
Faculty of Life Sciences, University of Manchester, Oxford road, M13 9PT, Manchester,
UK

The Sprouty proteins are intracellular regulators of receptor tyrosine kinase (RTK) signalling.
The family consists of 4 members (Sprouty1, 2, 3 and 4) which are conserved among
species, including zebrafish, Xenopus and mouse. In Xenopus, Sprouty1 and Sprouty2 have
been shown to repress the PLCγ/Ca++ pathway downstream of FGF, playing a role in the
regulation of the timing of morphogenesis during gastrulation. In mammals, Sprouty1, 2 and
4 inhibit the MAPK pathway downstream of FGF. In both systems Sprouty expression is
regulated by FGF, creating a feedback loop. Here we investigate the function of Sprouty3
during development which has not been addressed yet. We have found that, Sprouty3
expression starts at the end of gastrulation and at the tadpole stage it is restricted to the
spinal cord and the trigeminal nerve. This expression pattern overlaps with that of the
neurotrophin BDNF and its receptor TrkB, suggesting that it might modulate this pathway.
Sprouty3 morpholino-mediated knockdown in Xenopus tadpoles causes what appears to be
excessive branching in neurons of the spinal cord. To understand Sprouty3 function, I am
currently investigating whether Sprouty3 regulates any of the signalling pathways (MAPK,
PI3K/Akt or PLCγ/Ca++) downstream of BDNF. Moreover, I aim to address whether Sprouty3
has a role in axon guidance or neurite outgrowth in spinal cord neurons, by using
morpholino-mediated knockdown of Sprouty3 in Xenopus cultured spinal cord neurons.

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of
cardiac identity in first heart field derived cells

Susanne Gessert, Daniel Maurus, Thomas Brade, Paul Walther, Petra Pandur, Michael Kühl

Inst. for Biochemistry and Molecular Biology, Ulm University, Albert-Einstein-Allee 11, D-
89081 Ulm, Germany

DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain
superfamily of cell adhesion molecules and was recently suggested to be a target gene of
non-canonical Wnt signalling. During Xenopus embryogenesis, DM-GRASP is expressed in
cells of the first heart field (FHF) but not the second heart field (SHF), respectively. Loss of
DM-GRASP function results in smaller and malformed hearts. Injection of a DM-GRASP MO
did not affect specification of cardiac progenitor cells that will give rise to the FHF and SHF.
Later during development, expression of cardiac marker genes in the first heart field of
Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second
heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP
function resulted in defective cell adhesion as shown by electron microscopy. Additionally,
expression of DM-GRASP can rescue the phenotype that results from the loss of non-
canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling
are functionally coupled during cardiac development.

Ref.: Gessert et al., Dev. Biol., in press



                        Regeneration of the vasculature in Xenopus

                     Roberto Paredes, Nicholas Love and Enrique Amaya

The Healing Foundation Centre, Faculty of Life Science, University of Manchester,
Manchester M13 9PT, UK

Vascular regeneration, from a medical viewpoint, is an especially interesting phenomenon to
study as it represents an instance of neo-angiogenesis or neo-vasculogenesis. We have
begun to develop tools to analyze vascular regeneration in Xenopus tadpoles, using several
transgenic lines that label the vasculature in living tadpoles, such as the Tie2GFP and
Flk1GFP (VEGFR2GFP) lines. During the course of this work, we have discovered that the
Tie2GFP transgenic line marks a subset of cells in the circulation, in addition to the
endothelial cells of the vasculature. These labelled cells are also present within tissues and
are highly migratory. Their behaviour resembles that seen in migratory myeloid cells, and
indeed, these cells respond to injury by migrating quickly and efficiently to wounds. The Flk-
1GFP transgenic line also marks the vasculature and two other populations of cells: one in
circulation and another within tissues. By using FACS analysis we have found that the
circulating Flk1 positive cells do not express lymphocytes markers, such a CD8 and CD5,
suggesting they belong to the monocytic lineage. We are now beginning to investigate
whether these extra-vascular Flk1 and Tie2 positive cells contribute to the regeneration of
the vasculature in tadpoles.

System Properties Of The BMP4 Synexpression Group

Malte Paulsen, Emil Karaulanov, Christof Niehrs; Division: Molecular Embryology, German
Cancer Research Center, Heidelberg

The conserved BMP4 synexpression group has been described in various chordates,
constituting a system of intra- and extra-cellular positive and negative feedback loops.
Although the biochemistry of BMP signaling is largely known, the system properties of the
BMP4 synexpression group have not been studied in detail. Our work focuses on the role of
the intracellular negative feedback loop, consisting of Smad6, Smad7 and BAMBI, in BMP4
signaling. Loss-of-function experiments in Xenopus laevis embryos reveal that interference
with the negative feedback loop induces a higher variability in tail length and marker gene
expression. Real-time analysis of BMP4 signaling using a BMP-reporter cell line indicates
that the intracellular feedback loop induces a biphasic kinetic of BMP4 signaling. SiRNA
mediated knock down of BAMBI abolishes this biphasic signaling cascade and interferes with
the cell’s ability to differentiate between moderate and high concentrations of BMP4. These
findings indicate the requirement of the negative feedback loop for correct BMP4 signaling
kinetics, promoting robustness, and for correct response to a BMP4 gradient.


Using Xenopus to functionally define candidate genes for neonatal diabetes
Esther J. Pearl1, Constantin Polychronakos2, Marko E. Horb1
1
  Laboratory of Molecular Organogenesis, Institut de Recherches Cliniques de Montréal,
2
  Department of Pediatrics and Human Genetics, McGill University Health Center (Children’s
Hospital)
Neonatal diabetes affects infants in their first year of life, and the (usually monogenic)
etiology is known in only half of the cases. Homozygosity mapping in a family with unknown
etiology implicated a region on human chromosome 6 as potentially involved in neonatal
diabetes. One of the genes in this region has been selected for further study. This gene
codes for a new transcription factor that has not been well characterized, nor been shown to
be involved in either diabetes or pancreas development. In situ hybridization shows that it is
expressed in the endoderm during late neurula stages; at later stages it is expressed in the
developing dorsal and ventral pancreatic buds and in the developing gut. Because of its
pattern of expression we chose to pursue functional knockdown and overexpression studies
on this gene. Endoderm-targeted morpholino knock down resulted in abnormal foregut
development and decreased insulin expression. In contrast, we found increased insulin
expression in mRNA injected tadpoles. In conclusion, following on from homozygosity
mapping in humans we identified the Xenopus homologue of one candidate gene that
encodes a new transcription factor, and our preliminary functional studies indicate that it is
involved in early specification of the pancreas.



The lampbrush chromosomes of Xenopus tropicalis: identification and distribution of
RNA Pol III sites
May Penrad-Mobayed, Anwar El Jamil, Rasha Kanhoush and Caroline Perrin
“RNA/protein interactions and sex differenciation” group . Institut Jacques Monod; UMR
7592, CNRS-Universités Paris 6 et 7. 2, place Jussieu, 75251 Paris cedex 05, france.

The amphibian Xenopus tropicalis, whose genome has been recently sequenced, has
become an important model organism for vertebrate developmental genetics. Thus the
development of cytogenetic tools in this new model organism should contribute to an
understanding of the organisation of the amphibian genome and the mapping of a variety of
loci of interest. Lampbrush chromosomes found in the oocytes of many species are
particularly useful for the localisation of genomic sequences expressed during oogenesis. In
situ hybridization of specific probes to the nascent transcripts of lateral loops consistently
yield strong signals because of their hybridization to the many close-packed RNA transcripts
in these loops. For this purpose, we have constructed a working map of X. tropicalis
lampbrush chromosomes in which the ten bivalents of the oocyte karyotype can be identified.
We have also studied by immnofluorescence the distribution of targeting RNA Pol III subunits
in the nucleus, using three anti-pol III antibodies (R.G. Roeder). As was the case for X. laevis
(Murphy et al, 2002), the staining patterns are specific for each chromosome and constitute a
supplementary tool for their identification. Lampbrush chromosomes also offer a high
resolution approach for analysis of chromosome heteromorphism. Like many other
amphibians, Xenopus belongs to the genetic system of sex determination (ZZ/ZW) and has
poorly differentiated sex chromosomes, which are undistinguishable in the mitotic karyotype
Our hope is that this working map will allow the identification of the Z and W sex
chromosomes.



A mutagenesis screen in Xenopus tropicalis

Isabelle Philipp, T. Blair Gainous, Toral B. Trivedi, Hyeyoung Chung, Mustafa K. Khokha,
Timothy C. Grammer and Richard M. Harland
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA
94720-3200
Mutagenesis screens are an efficient strategy to identify genes required in development.
Many genes with important developmental functions have been discovered through mutant
phenotypes. However, we are still far from a thorough understanding of the genetic control of
vertebrate embryogenesis. Xenopus laevis has historically been an important model system
to elucidate mechanisms of development using classical embryology and biochemistry, but
because of its long generation time and its pseudotetrapleudy, has not been a feasible
genetic model. On the other hand, the related species Xenopus tropicals is diploid, has ten
chromosomes, a shorter generation time and shares all the technical advantages of its larger
cousin. We are performing a genetic screen to unveil more of the genes that are required for
vertebrate development in Xenopus tropicalis. Pilot screens with Gamma radiation and ENU
in Xenopus tropicalis, which generated approximately 500 F1 frogs, were screened for
mutant phenotypes via gynogenesis. So far, we have identified several mutant phenotypes
and are in the process of mapping the mutations. While managing mutant lines and mapping
the responsible mutant genes remains so far a laborious and tedious task it is still more
feasible then in other vertebrates with larger chromosome numbers.
One of these mutants, blimpy, shows a cardiovascular phenotype. This mutation was
mapped to one of the ten linkage groups. We are currently determining the gene behind this
mutation, as well as performing a more detailed characterization of the phenotype. Other
mutations identified show axial defects (egghead-1, egghead-2, hourglass-like, kopflos) or
mis-development of the eye (lumpy eye). The phenotypes we identified so far reinforce the
value of expanding a forward genetic approach to Xenopus tropicalis in order to identify new
genes involved in vertebrate development.


Origin and compartmentalization of placodes in Xenopus laevis revealed by fate map
analysis

Mareike Pieper, Gerhard Schlosser
Brain Research Institute, University of Bremen, PO Box 330440, D-28334 Bremen (e-mail:
pieperma@uni-bremen.de)

Placodes are specialized regions of the ectoderm and together with neural crest cells they
give rise to sense organs and neurons of the vertebrate head. These placodes become
visible as local thickenings during neurulation. Several types of placodes can be
distinguished based on their fate and position: adenohypophyseal, otic, olfactory, trigeminal
and profundal, lens, a series of epibranchial and a series of lateral line placodes. The
expression patterns of several genes like Six1 and Eya1 suggest that all ectodermal
placodes arise from a common ectodermal precursor region (panplacodal primordium)
localized around the anterior neural plate. Six1 and Eya1 are strongly expressed in the
panplacodal primordium and later on in all placodes. They may play an important role during
development of different placodes. To verify that all placodes arise from a common precursor
we generated a fate map of the panplacodal primordium in neural plate stage Xenopus laevis
embryos. We injected fluorescent dyes into small groups of cells within the panplacodal
primordium and defined the marked placodes in tailbud stage Xenopus laevis embryos. Our
fate map analysis confirms that all ectodermal placodes arise from a common precursor and
indicates their region of origin. We further filmed cells of the deep ectodermal layer in the
region of the panplacodal primordium from neural plate stage on to create time-lapse movies.
These movies allow us to follow cell movements and cell divisions to elucidate which
processes contribute to the compartmentalization of the panplacodal primordium into
individual placodes.


Groucho-related co-repressors mediate the interaction of FGF and Wnt signaling
M.E. Pownall, Biology Department, University of York, YO10 5YW, United Kingdom
Interactions between the FGF and Wnt signalling pathways underpin a number of
developmental processes, including mesoderm formation in Xenopus embryos. However,
the nature of the interactions between these key signalling pathways remains unclear. Here
we find that the transcriptional co-repressor Groucho-related 4 (Xgrg4/TLE4) is
phosphorylated downstream of the FGF signal transduction pathway. We show that this
modification reduces the ability of Xgrg4 to inhibit transcriptional targets of canonical Wnt
signalling and that this is dependent on a consensus site for MAPK phosphorylation in Xgrg4.
These data present us with a novel molecular mechanism where FGF and Wnt signalling
pathways synergise at the level of the co-repressor Xgrg4: the weakening of Xgrg4
repression by FGF signalling combined with the stabilization of b-catenin by Wnt signals
leads to robust output from Wnt target genes.



Differential regulations of quercetin and its glycosides on ligand-gated ion channels



Mi Kyung Pyo, Byung-Hwan Lee, Sun-Hye Choi, Tae-Joon Shin, Sung Hee Hwang, Sang-
Mok Lee, and Seung-Yeol Nah

Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-
701 Korea


Quercetin, one of the flavonoids, is a compound of low molecular weight found in various
plants and shows a wide range of diverse neuropharmacological actions. In fruits and
vegetables quercetin naturally exists as monomer- (quercetin-3-O-rhamnoside), dimer- (rutin)
or trimer-glycosides [quercetin-3-O-(2G-rhamnoside)] at carbon-3. In the previous studies, we
demonstrated that quercetin inhibits both glycine and 5-HT3A receptor channel activities
expressed in Xenopus oocytes. However, the roles of carbohydrate attached at carbon-3 of
quercetin on glycine and 5-HT3A receptor channel activities are not well studied. In the
present study we investigated the effect of quercetin glycosides on the human glycine α1
receptor and mouse 5-HT3A receptor channel activities expressed in Xenopus oocytes using
a two-electrode voltage clamp technique. In oocytes expressing glycine receptors,
application of quercetin and its glycosides reversibly inhibited glycine-induced current (IGly) in
order of quercetin > rutin > quercetin-3-O-rhamnoside > quercetin-3-O-(2G-rhamnoside). In
oocytes expressing 5-HT3A receptor, quercetin’s and its glycosides’ inhibitions on IGly and I5-HT
was dose-dependent. Application of quercetin and its glycosides reversibly inhibited 5-HT-
induced current (I5-HT) in order of quercetin-3-O-(2G-rhamnoside) ≥ quercetin > rutin >
quercetin-3-O-rhamnoside. The inhibition of IGly by quercetin glycosides was not competitive,
whereas the inhibition of I5-HT by quercetin glycosides was competitive manner. These results
indicate that glycosylations of quercetin affect on glycine receptor channel activity, whereas
glycosylations of quercetin did not affect on 5-HT3A receptor channel activity. These results
indicate that quercetin glycosides might regulate the human glycine α1 and mouse 5-HT3A
receptors with differential manners.


Does Sulf1 affect how Sonic diffuses during neurogenesis?
S.A. Ramsbottom and M.E. Pownall
Department of Biology, University of York

Heparan sulphate proteoglycans (HSPGs) are large molecules distributed ubiquitously, both
at the cell surface and within the extracellular matrix. These molecules are known to play
essential roles in developmental cell signalling and the differential sulfation of HSPG chains
gives rise to much variability in their binding specificity. Sulf1, an N-acetlyglucosamine O-6
endosulfatase, specifically removes sulphate groups from HSPG chains in regions of high
sulfation, and our lab has previously shown that Sulf1 activity leads to the attenuation both
BMP and FGF signalling.

The expression profile of Sulf1 within the neural tube is similar to that of Sonic Hedgehog
and work in chick has shown that Sulf1 is able to increase the concentration of Sonic at the
cell surface in a cell autonomous manner (Danesin et al., 2006). Due to the fact that HSPG
chains are required for the diffusion of Sonic through of field of cells (The et al., 1999), Sulf1
provides a good candidate as a regulator of Sonic distribution and activity.

I have characterised the expression of XtSulf1 and XtSonic during open neural plate and
neural tube stages of primary neurogenesis. The expression of molecular markers of
specific neuronal precursors have also been analysed at these stages, including Nkx6.1,
Dbx, Lbx1 and Isl1. Overexpression of both Sulf1 and Sonic changes the spatial distribution
of these markers indicating that Sulf1 may be facilitating either the movement or activity of
Sonic hedgehog during primary neurogenesis.


     The Interaction of the Wnt and Nodal Pathways during Organizer Formation in
                                       Xenopus laevis
                            Christine Reid and Daniel S. Kessler
                                 Dept. of Cell and Dev. Bio.
                       University of Pennsylvania School of Medicine
                                   Philadelphia, PA 19104

The Wnt and Nodal pathways are known to interact in a variety of different tissues as well as
in the maintenance of many different types of stem cells. Within the developing Xenopus
embryo, the Wnt and Nodal pathways overlap in the area of the Spemann Organizer, a
region of tissue on the dorsal side of the embryo essential for embryonic patterning and axis
formation. Both the Wnt and Nodal pathways are essential for organizer formation, yet little is
known about how these pathways interact at the level of target gene promoters to affect
organizer gene expression. Goosecoid (Gsc) is an organizer specific gene with a well-
defined promoter containing both a Wnt responsive element and a Nodal responsive
element. Expression of Wnt pathway effectors Siamois (Sia) and Twin (Twn) with Nodal
leads to synergistic activation of the Gsc promoter. We hypothesize that this synergistic
activation seen is due to the recruitment of a common co-factor among the Wnt and Nodal
effectors. Chromatin immunoprecipitation has revealed that Wnt effectors Sia and Twn and
Nodal effectors Fast-1 and Smad2 bind to the endogenous Gsc promoter. A candidate
approach was taken to identify potential co-factors. Inhibition of p300 activity abrogates both
Sia/Twn and Nodal mediated activation of Gsc expression, suggesting that Wnt and Nodal
effectors are recruiting p300 as a co-factor. Current work focuses on establishing the
relationships among Wnt and Nodal pathway effectors and determining whether recruitment
of p300 is essential for Sia/Twn or Fast-1/Smad2 mediated activity during organizer
formation. Defining the mechanism by which Wnt and Nodal pathway effectors function to
promote the formation of the organizer is critical to understanding how the overlap of two
distinct signaling pathways cooperate to establish the organizer.

Mapping X. tropicalis Mutations with Gynogenesis and Centromeres
Michael J. Reilly1, Vladimir Krylov2, Jaroslav Macha2, Lucie Tumova2, Tereza Tlapakova2,
Dipankan Bhattarachaya3, Joanne Cheung3, Sarah Kaufmann3, Dang Lam3, Catherine Ngo3,
Neha Prakash3, Phillip Schmidt3, Toral Trivedi3, Anita Abu-Daya1, Timothy Geach1, Elisenda
Vendrell1, Holly Ironfield1, Timothy C. Grammer3, Mustafa Khokha4, Richard Harland3, and
Lyle Zimmerman1
1
  National Institute for Medical Research, London UK
2
  Department of Cell Biology, Charles University, Prague, Czech Republic
3
  Department of Molecular & Cell Biology, University of California, Berkeley USA
4
  Yale University School of Medicine, New Haven, Connecticut USA

Forward genetics in X. tropicalis will rely on efficient mapping of identified mutations. Rapid
low-resolution mapping of recessive loci can be accomplished using a small number of
gynogenetic embryos derived from an individual female heterozygous for the mutation. A
high proportion of recessive phenotypes uncovered in such embryos, regardless of their
chromosomal location, will appear linked to centromeric polymorphisms on the
corresponding chromosome; a panel of 10 centromeric markers can therefore be used to
assign mutations rapidly to one of the 10 X. tropicalis linkage groups. To identify centromeric
polymorphisms for use in a rapid mapping panel, we again used gynogenesis. Polymorphic
loci will only appear heterozygous in individual gynogenetic embryos when a crossover has
occurred between the locus and its centromere, so heterozygotes are rare at centromeric
loci. We have used this approach to identify polymorphic markers within ~1.5cM of all 10
centromeres. For each locus, >48 individual gynogenetic embryos were analyzed for
candidate Simple Sequence Length Polymorphisms (SSLPs); centromere-linked loci gave
<2/48 heterozygous gynogenetic embryos. Using cDNA probes obtained from the candidate
centromere region, Fluorescent In Situ Hybridization coupled with a tyramide amplification
step (FISH-TSA) independently confirmed cytological centromere locations. The centromere
marker panel is now being used to place mutations on the genetic map.


The Siggi(KIAA0888) homolog of X. tropicalis is essential for early embryogenesis.
#Torrejón M, *Gupta R and *Reinsch S. #Department. of Biochemistry. and Molecular.
Biology., University. of Concepción, Chile. *NASA-Ames Research. Center, Moffett Field CA.
USA

We generated several mutant lines in X. tropicalis using green fluorescent protein (GFP) as a
gene-trap. A founder animal with an insert in the Siggi gene (humanKIAA0888) was identified
using 5'RACE. This is a developmentally normal female. Several matings of this founder
generated hundreds normal heterozygote tadpoles that matured. GFP expression was strong
throughout early development in the CNS, and craniofacial cartilage and weak in leg bones
of metamorphosing tadpoles. GFP expression in the eye is visible in adults. Matings between
heterozygote transgenic F1 animals to generate homozygote F2 yielded hundreds of
embryos of which the homozygotes presented stronger GFP expression. Homozygotes were
significantly smaller than clutchmate heterozygotes and failed to thrive; all died within 2
weeks. A transcript cloned from St44 wild-type tadpoles is predicted to encode a 79kDa
protein. Several altered transcripts were cloned by RT-PCR from transgenic animals
indicating that a duplication of at least 1 exon has occurred. In situ analysis shows
expression in the developing CNS, neural crest and muscle. Morpholino injection into early
embryos gives a dose-dependent response. High concentrations cause developmental delay,
exogastrulation and axial defects. Intermediate concentrations allow development to later
stages but all of them present axial defects. A low concentration gives phenotypically normal
tadpoles that show later defects in development and a failure to thrive similar to homozygote
F2. This growth reduction can be rescued by co-injection of RNA encoding the full-length
predicted protein. However, the injected RNA does not rescue early defects. Finally, RNA
encoding the full-length protein fused to GFP shows nuclear localization and the
overexpression also causes an abnormal phenotype. The KIA0888 predicted protein has no
significant homology with known proteins and is an Intrinsically Disordered Protein

     5-HT2B-mediated serotonin signalling participates in retinal and craniofacial
               morphogenesis during Xenopus laevis development
                  Elisa Reisoli1, Michela Ori1, Stefania De Lucchini2, Irma Nardi1
  1
      Unità di Biologia Cellulare e dello Sviluppo, Dipartimento di Biologia, Università di Pisa,
                                               Pisa, Italy
                               2
                                 Scuola Normale Superiore, Pisa, Italy

    Serotonin (5-HT) is a neurotransmitter that mediates a wide variety of effects in the central
and peripheral nervous system. Experimental evidences demonstrated that serotonin even
has an important role as growth and differentiating factor for neuronal and non-neuronal cells
by controlling proliferation, migration and apoptosis during development. All the biological
actions of 5-HT are mediated by G-coupled receptors and, among these, 5-HT2B receptor is
expressed during CNS, heart and craniofacial development. By using Xenopus laevis as a
model system, we demonstrated that 5-HT2B receptor loss of function determines a
decrease in the proliferation rate of retinoblasts and increases the apoptosis of retinal cells
thus resulting in abnormal eye morphology. In order to further investigate the 5-HT2B role
during development, we performed complementary experiments of gene gain of function.
The 5-HT2B over-expression, leads to the formation of eyes with irregular form, position and
orientation and showing defects in the optic fissure closure and in the pigmented epithelium
formation. A detailed molecular analysis revealed a disorganization of the typical laminar
retinal structure and the presence of differentiated retinal cells in ectopic position. As
pharmacological treatments with 5-HT2 antagonists elicited in mice craniofacial alterations,
we are now studying the formation of craniofacial skeletal elements and their associated
muscles in 5-HT2B gain and loss of function experiments. In particular, 5-HT2B gene gain of
function results in altered formation of the jaw cartilage and correlated muscles.
    Conclusion: We showed that 5-HT, via 5-HT2B receptor, is among the key extracellular
signals that control Xenopus retinal histogenesis and eye morphogenesis. Moreover, our
results suggest for the first time a direct involvement 5-HT2B receptors in mediating the
serotonin action on craniofacial morphogenesis by influencing the formation of skeletal
elements and that of the connected muscles.




Novel insights for understanding host response to chytridiomycosis

Ribas L., Li M-S., Kroll J. S., Garner T. and Fisher M. C.

Dept of Infectious Disease Epidemiology. Imperial College London.


        Chytridiomycosis is an emerging infectious disease of amphibians caused by the
chytrid Batrachochytrium dendrobatidis (Bd). The disease has been associated with global
amphibian declines and has driven species to extinction. Pathogen occurs with a high
prevalence in many farmed and laboratory species of amphibians and is being rapidly
dispersed worldwide. Last March, the World Animal Health Organisation (OIE) has
recognized Bd for its serious nature of infection. Although recent epidemiological studies
have linked the spread of this disease to climate change, altitude levels and other local
factors, the principle drivers responsible for the emergence of chytridiomycosis remain
unclear. In our lab, we have housed Xenopus tropicalis, a high resistant species, at warm
(26oC) and cold (18oC) temperatures subjected at repeat-bathed Bd infections. Results
showed that X. tropicalis decreased its infection level at day 42 even when facing a
persistent 12 day cycle of reinfection, demonstrating the generation of a potent anti-Bd
response. In order to more widely describe the host response to infection by Bd, we utilised a
high-density oligonucleotide microarray platform. Here we present novel and preliminary data
showing how X. tropicalis mounts a potent, temperature dependent, response to Bd by
regulating significantly-different transcriptional profiles. We also studied in deep several
immune gene expression patterns by using qPCR. Our analyses demonstrated that Bd
infected animals developed an activation of several molecular and cellular processes,
including protein biosynthesis, cell division, cell structure, signal recognition, metabolic
pathways and immune response to challenge and clear Bd infection.




Mix.1/2-dependent control of FGF availability during gastrulation is essential for
pronephros development in Xenopus.
Alexandre Colas*, Jérôme Cartry*, Isabelle Buisson*, Muriel Umbhauer*, James C. Smith‡,
Jean-François Riou*
*
  UPMC Univ Paris 06, CNRS, Laboratoire de Biologie du Développement UMR 7622, 9 quai
Saint-Bernard, 75005 Paris, France
‡
  Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

Although FGFs are known to affect mesoderm patterning in the marginal zone, their
influence on intermediate mesoderm specification during gastrulation is ignored. Here, we
show that pronephros precursors express Xbra and thus are likely exposed to FGF. Yet,
overactivation of FGF leads to an inhibition of the nephrogenic mesoderm markers XPax-8
and Xlim-1 at early neurula stage, indicating that a strict control of FGF signals is necessary
to allow pronephros specification. We provide evidence that this control is mediated by the
paired-like homeobox genes Mix.1 and Mix.2. Morpholino-based Mix.1/2 knockdown, or
repression of Mix.1 target genes with an enRMix.1 construct, causes an expansion of FGF4
and FGF8 expression in lateral marginal zone at gastrula stage, together with an inhibition of
pronephros development at neurula and tailbud stages. This does not result from an indirect
effect of Mix.1/2 knockdown on the organizer. Neurula expression of Xlim-1 and XPax-8 can
be rescued in Mix.1/2 morphants by intrablastocœlic injections of the FGFR inhibitor SU5402
at mid-gastrula stage, showing that inhibition of pronephros development results from an
increase of FGF signalling. We further show that Mix.1 overexpression results in the down
regulation of FGF3, 4, 8 and XmyoD, in addition to Xbra. However, cells overexpressing
Mix.1 can normally populate somites, showing that Mix.1 does not affect their fate cell
autonomously. These data support the idea that Mix.1/2 play a critical function in the
regulation of levels and/or duration of FGF signals to which pronephros precursors are
exposed in the marginal zone during gastrulation.


Comparative study of immunologically-relevant genes in Xenopus laevis and Silurana
(Xenopus) tropicalis
Jacques Robert, Hristina Nedelkovska, Heidi Morales, Ana Goyos, Jessica Sowa, and
Seema Chida
Department of Microbiology & Immunology, University of Rochester Medical Center
Rochester, NY 14642, USA

    The accessibility of early developmental stages devoid of maternal influence and
metamorphosis makes Xenopus an excellent comparative model to study T cell
differentiation during ontogeny. Moreover, tadpoles have CD8 T-cells despite suboptimal
expression of classical MHC class Ia (class Ia) molecules, which are necessary for CD8 T-
cell differentiation. We have characterized in X. laevis a novel non-classical MHC class Ib
(class Ib) gene XNC10, whose expression is tightly associated with the CD8 T-cell lineage
during thymocyte differentiation since early in ontogeny.
   We are postulating that XNC10 contributes to CD8 T-cell differentiation in class Ia-
deficient larvae. To further reveal the role of class Ia and class Ib gene products in CD8 T-
cell differentiation, we are developing transgenesis strategies to impair their expression in
vivo by RNAi knockdown using MHC-defined isogenetic clones. We have targeted b2-
microglobulin (β2-m) that is required for surface expression of both class Ia and XNC10. We
have produced transgenic clones expressing an anti-b2m shRNA using the Sleeping Beauty
transposase. Preliminary results suggest that the shRNA expression is not deleterious in vivo
and significantly knocks down β2-m. Additionally, all transgenic offspring should carry the
transgene since they will be produced by gynogenesis.
   The rapid evolution rate and poor conservation of class Ib genes between vertebrate
species is intriguing. We have evaluated the degree of class Ib gene conservation between
X. laevis and S. silurana thanks to its available genome sequence. We have identified 29
putative class Ib genes, 11 of which are supported by expression data (including several
preferentially expressed in lymphoid tissues). Phylogenetic analysis supports homology of
only 5 of the 11 X. laevis class Ib subfamilies with S. tropicalis class Ib genes. Reagents
(primers, antibodies) are now needed to further characterize functionally equivalent class Ib
genes in S. tropicalis.
Research Support: NIH: F31-AI68610-01 (A.G.), T32-AI 07285 R01-CA-108982-02 (H.N;
H.M.), R24-AI-059830-01 and NSF: MCB-0136536




FGF-mediated posterior neural patterning requires Dazap2

Daniel D. Roche1, *, Karen J. Liu2, *, Richard M. Harland3, Anne H. Monsoro-Burq1, 4, 5
1
 Institut Curie, Orsay, France; CNRS, UMR146, Orsay, France; 2 Department of Craniofacial
Development, King's College, London, UK; 3 UC Berkeley, MCB Department, USA; 4 Collège
de France, Paris, France.

The organization of the embryonic neural plate requires coordination of multiple signal
transduction pathways, including fibroblast growth factors (FGFs), bone morphogenetic
proteins (BMPs), WNTs and retinoids. Many studies have suggested that a critical
component of this process is the patterning of posterior neural tissues by an FGF-caudal
signaling cascade. Here, we have identified a novel player, Dazap2, and show that it is
required in vivo for posterior neural fate. Loss of Dazap2 in embryos resulted in diminished
expression of hoxb9 with a concurrent increase in the anterior marker otx2. Furthermore, we
found that Dazap2 is required for FGF dependent posterior patterning; surprisingly, this is
independent of cdx4 activity. Functionally, we found that increasing Dazap2 levels can
suppress head formation, expand the neural plate and induce posterior neural markers. This
activity overcomes the anteriorizing effects of noggin, and is downstream of FGF receptor
activation. Our results strongly suggest that Dazap2 is a novel and essential branch of FGF-
induced neural patterning.

Heat-shock inducible recombinases in transgenic Xenopus laevis

Magdalena Roose, Kathrin Sauert, Gerhart U. Ryffel, Gülüzar Turan, Christoph Waldner
Institut für Zellbiologie (Tumorforschung), Hufelandstr. 55, D-45122 Essen, Germany

The transcription factor HNF1β plays a prominent role in Xenopus pronephros development
and heterozygous mutations in humans are associated with maturity onset diabetes of the
young (type five), defective kidney development, genital malformation and decreased
pancreas size. To identify the critical time window for HNF1β function in development we use
the Cre/loxP and the FLP/FRT system in a transgenic approach. Cre and FLP recombinases
were made inducible by placing them under the control of the Xenopus heat-shock promoter
(HSP) and thus can be activated by heat-shock at a defined time-point. We generated
founder animals for both recombinases. To test the activity of these recombinases, founder
animals were crossed to a reporter strain expressing a Cre- or FLP- inducible yellow
fluorescent protein. Using this reporter strain we were able to identify two inducible Cre
strains (HSPCre1 and HSPCre13). Notably, HSPCre13 contains in addition a gene encoding
the red fluorescent protein tdTomato that can be used in crossings as marker for the
presence of the recombinase. Successful heat-shock induction was obtained from the
blastula to the tailbud stage. PCR analysis revealed that recombination starts within the one
hour heat-shock period and is essentially complete within 4 hours. Yellow fluorescence could
be observed as early as 6 hours after heat-shock. We also crossed the HSPCre1 strain to an
effector strain carrying an inducible derivative of HNF1β under the control of the CMV
promoter. Upon heat-shock at the gastrula stage edemas, reduced pronephros formation and
malformation of the gut were observed. For a more restricted overexpression of HNF1β we
will also use strains with a tissue restricted effector gene or by applying local heat-shocks
using laser beams.


Timing of neuronal differentiation and patterning of vertebrate telencephalon by the
transcription factor FoxG1

Martin Roth and Nancy Papalopulu
University of Manchester, The Michael Smith Building, Oxford Road, Manchester M13 9PT

In mammals, FoxG1, a member of the forkhead family of winged helix transcription factors, is
required to maintain neural progenitors and to program sequential neuronal fate during
cortical neurogenesis. In Xenopus overexpression assays, FoxG1 is remarkably bifunctional
in that it induces ectopic neurogenesis when overexpressed at low doses and suppresses
endogenous neurogenesis when the dose is increased as little as two-fold. We report that
co-expression of the transcritptional co-repressor groucho-3 is sufficient to convert a low
dose FoxG1 phenotype to a high dose one, suggesting that the activity of FoxG1 in the
nucleus is regulated by the presence of co-factors.
There are four Xenopus grouchos with partially overlapping expression patterns in the
developing anterior neural plate and telencephalon, however not all grouchos showed a
functional interaction with FoxG1 in our neurogenesis assays. Interestingly groucho-3 is co-
expressed with FoxG1 in the anterior neural plate, when neurogenesis is suppressed and is
down regulated in the tadpole forebrain when neurogenesis commences. We aim to address
whether the activity of endogenous FoxG1 is regulated by groucho-3 and the effect of this
interaction on neurogenesis.
.


PPARbeta controls Xenopus Nodal Related genes during gastrulation

Nicolas Rotman, Francesco La Spada and Walter Wahli

Center for Integrative Genomics, National Research Center Frontiers in Genetics, University
of Lausanne, CH-1015 Lausanne, Switzerland


       The peroxisome proliferator-activated receptors (PPARs) constitute a sub-group of
the nuclear receptor family that comprises three isotypes, alpha, beta and gamma. PPARs
 are key regulators of glucose and lipid homeostasis and have been shown to play a role in
 inflammation and wound healing. In addition, they also control important developmental
 processes in human and rodents. Indeed, PPARs play a role in placenta formation,
 adipogenesis, differentiation of the skin and intestinal epithelia and may also act in neural
 development. However, it is likely than the requirement for PPARs in early development is
 broader and should therefore be investigated in more details. We have undertaken an in
 depth analysis of the role of PPARs in Xenopus laevis, focusing first on PPARbeta, which is
 abundant all through early embryogenesis.
         Down regulation of PPARbeta by the mean of morpholino injection has dramatic
 effects on both gastrulation and early organogenesis: PPARbeta morphants show very
 reduced elongation of the antero-posterior axis and present a plethora of differentiation
 defects affecting the three germ layers. Time course analyzes combined with region specific
 knock-down of PPARbeta indicate that it controls gastrulation movements. At the molecular
 level, PPARbeta regulates the Nodal pathway by controlling the transcription of the 6 Nodal
 ligands (Xenopus nodal related genes 1-6; Xnr 1-6). In addition to knock down experiments,
 over-expression and over-activation of PPARbeta were also conducted that confirm the
 central role of that nuclear receptor in the regulation of Xnr genes. Interestingly, PPARbeta
 behaves as a positive regulator of Xnr genes immediately prior to gastrulation but functions
 as a repressor during gastrulation.
 All together, our data pinpoint PPARbeta as a key factor in early embryo patterning. Protein-
 protein interaction studies and ChIPs are ongoing that should help to better understand the
 molecular basis of Xnr gene regulation by PPARbeta.




Role of cell polarity in cell fate determination during Xenopus primary neurogenesis
Sabherwal N1, Chalmers AD2 and Papalopulu N1.
1. Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road,
Manchester M13 9PT; U.K. 2. Centre for Regenerative Medicine, University of Bath, Bath BA2
7AY, U.K.

The neuroectoderm in Xenopus is a bilayered epithelium, consisting of outer layer of apico-basally
polarized cells and an inner (deep) layer of non-polar cells. Only deep cells give rise to primary
neurons at the neural plate stage. Whether the polarity of cells affects their fate during
neurogenesis is not known.

We report the results of differential over-expression of polarity components on cell fate. Over-
expression of Lgl2 abolishes the polarity of outer cells by disrupting their apical domain and tight
junctions but does not lead to extra neurons. Overexpression of the bHLH proneural gene X-Ngnr-
1 results in ectopic neurons in the deep layer only, but its overexpression in combination with Lgl-2
results in ectopic neurons located in an intermediate position between deep and superficial layers.
Lineage tracing and time-lapse microscopy clearly demonstrate that outer cells with disrupted
polarity due to Lgl2 over-expression get internalized and this accounts for their “intermediate”
position. Thus Lgl-2 changes both the position and neuronal competence of outer cells.

Conversely, over-expression of aPKC or its kinase domain (aPKC-CT) leads to hyperapicalized
superficial cells but did not significantly reduce the primary neurons (shown by N-tubulin
expression). Interestingly, over-expression of a membrane targeted version of aPKC, or the
nuclear version of aPKC-CT reduced the N-tubulin expression. On overexpression of the
membrane tethered aPKC, tagged aPKC is found in the nuclear extract of cells. Work is in
progress to address how this differential subcellular localization and kinase activity of aPKC affects
cell fate.
Krüppel-like factor-2 and Ets transcription factors physically interact during vascular
development.

Matthew C. Salanga, Stryder M. Meadows and Paul A. Krieg
Department of Molecular, Cellular and Developmental Biology, The University of Arizona

Krüppel -like factors (Klfs) comprise a multi-gene family of transcription factors distinguished
by a highly conserved zinc finger DNA-binding domain. Klfs bind a CACCC nucleotide motif
and may repress or activate gene transcription depending on the particular Klf protein or the
promoter context. Ets transcription factors comprise another multi-gene family of transcription
factors, which bind a GGAA/T consensus site. Subsets of factors from both families are
expressed in the vasculature of the early embryo, and therefore may play a role in embryonic
vascular development. Transgenic analysis of Flk-1 indicates that both Klf and Ets sites in
the first intron enhancer are required for efficient transgene expression, and therefore
embryonic vascular development (see also Meadows et al.). We have demonstrated that the
endothelial expressed factor Klf2 can form a complex with the Ets family proteins Erg or Fli,
which are also expressed in endothelial cells. Follow up studies will use chromatin
immunoprecipitation methods to determine whether Klf and Ets proteins bind directly to the
Flk-1 enhancer in vivo. We are investigating the expression profiles of all Klf family members
in the developing embryo, with the rationale of identifying additional Klf-Ets relationships.




Title:         Deciphering the role of the Anterior Endoderm in cardiac specification
Authors:       Lee Samuel & Branko Latinkić
Insitution:    Department of Genetics, School of Biosciences, University of Wales Cardiff,
               Museum Avenue, Cardiff, UK, CF10 3US

The heart is the first functional organ of vertebrate embryogenesis, however little is known
about the early specification events of cardiogenesis. Evidence in the chick and amphibian
suggests a requirement for the Anterior Endoderm (AE) in cardiac induction to direct
mesoderm toward a cardiac fate.

We have devised a model testing the cardiac inducing activity of the AE by addressing its
ability to re-specify pluripotent embryonic ectoderm. Conjugation of early gastrula AE to
Animal Cap (AC) cells results in robust expression of cardiac differentiation markers and
formation of contractile tissue. Lineage tracer analysis has revealed that the cardiac tissue is
only induced in the AC, and thus the AE is sufficient to induce cardiogenesis, with signals
emanating from the AE specifying cardiac fate.

Further characterisation has revealed that the AE loses its inductive properties toward the
end of gastrulation, and we have revealed that its inductive properties are restricted, with no
induction of skeletal muscle or neural markers.

Using this model, we have been investigating the early signalling and mechanisms of
cardiogenesis. We have manipulated signalling pathways in our model in attempt to define
the early inductive events. Our results have revealed an involvement for Nodal and FGF in
the specification and subsequent expansion of the cardiac precursors, in addition to an
absolute requirement for low Wnt signalling. On going analysis is directed towards
elucidating the exact timing and sequence of events required for cardiac induction.
Smad4 signaling in early Xenopus development

Veronika Sander, Hadrien Demagny, Vincent Taelman and Eddy M. de Robertis
Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA
vsander@mednet.ucla.edu

Recently, our laboratory described that the activity of the transcription factor Smad1 is
downregulated by MAPK (activated by FGFR and IGFR) and GSK3 phosphorylations. GSK3
activity is regulated by Wnt, thus explaining many of the ventralizing effects of Xwnt8 at the
gastrula stage in Xenopus (Fuentealba et al. Cell, 2007).
In the present study, we are investigating the physiologial effects of a single MAPK
phosphorylation site (PXSP) found in Smad4, the common co-factor of all receptor-activated
Smads, which is preceded by two GSK3 phosphorylation sites (SXXXS). We have devised a
biological assay, in which we perform single mRNA injections into specific blastomeres at the
16- or 32-cell stage of Xenopus embryos. Preliminary results indicate that Smad4 activity is
regulated by phosphorylation - similar to Smad1 – as phosphorylation-resistant mutants of
Smad4 are hyperactive. Embryos injected with hyperactive Smad4 mRNA form secondary
blastopores and tails. These phenotypes are observed more frequently when mutant Smad4
is co-injected with Smad3. If the GSK3 phosphorylation is regulated by Wnt, this would
represent another intersection point for the integration of cell-cell signaling.
In addition, we provide evidence for a role of Smad2 and Smad3 in the establishment of the
blastula preorganizer (or BCNE, blastula Chordin- and Noggin- expressing center). Upon
injection of Smad2/3 morpholinos we observed a reduction of Chordin expression at blastula,
indicating that TGFβ/Nodal/Activin signaling is required for dorsal gene activation at blastula
in addition to β-catenin.




Prohibitin 1 is a novel, essential factor in neural crest specification
Martina Schneider1, Doris Wedlich1, Alexandra Schambony2
1
  Zoological Institute II, Department of Molecular Developmental- and Cellphysiology,
University of Karlsruhe, Germany; 2Biology Department, Developmental Biology, Friedrich-
Alexander-University Erlangen-Nuremberg, Germany

Prohibitin 1 and Prohibitin 2 are evolutionary highly conserved proteins with multiple
functions in all eukaryotes. Both proteins are required for mitochondrial function and in
addition play a role in transcriptional regulation and cellular signalling. Prohibitin 1 is part of
the Ras/Raf signalling cascade, has been implicated in the regulation of apoptosis and cell
cycle, and modulates the activity of different transcription factors. We have identified and
cloned the Xenopus homologues of Prohibitin 1 and Prohibitin 2, XPhb1 and XPhb2. Here,
we report a novel role for XPhb1 in the regulation of neural crest development in Xenopus
laevis.
XPhb1 shows a marked specific expression in the neural folds, in the developing eye and in
the neural crest. Loss-of-function studies by an antisense Morpholino knock-down
demonstrated an essential role of XPhb1 in the specification of the neural crest. XPhb1 is
required for the upregulation of FoxD3, slug and twist, but not snail or AP2 downstream of c-
myc, demonstrating that XPhb1 is a novel essential component in neural crest development.


                Regulation of neurogenesis by Eya1 and Six1 in Xenopus
   Gerhard Schlosser1, Doris Völker1, Angelika Stammler1, Samantha Brugmann2, Mike W.
                                Klymkowsky3, Sally A. Moody2
1
  Brain Research Institute, University of Bremen, PO Box 330440, 28334 Bremen
(gschloss@uni-bremen.de), 2Dept. Anatomy and Regenerative Biology, Institute for
Biomedical Sciences, The George Washington University, Washington, DC 20037, USA,
3
  Dept. Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO
80309-0347, USA

At neural plate stages, transcription factors Eya1 and Six1 are strongly expressed in the pre-
placodal ectoderm. This region, which forms a crescent around the anterior neural plate,
subsequently forms various cranial placodes, which contribute to many sensory organs and
ganglia of the vertebrate head. Gain and loss of function experiments in Xenopus show that
Eya1 and Six1 synergistically act as essential regulators of placodal neurogenesis. After
morpholino-mediated knockdown of these genes, proliferation is compromised and placodal
expression of genes promoting neuronal progenitors (SoxB1 genes: Sox2, Sox3) or neuronal
differentiation (NeuroD, N-Tubulin) is reduced. Overexpression of Eya1 and Six1 reveals
dosage dependent effects. At high levels, Eya1 and Six1 promote proliferation and ectopic
SoxB1 gene expression in the non-neural ectoderm but block placodal neuronal
differentiation, while at lower levels they promote neuronal differentiation in placodes
downstream of and/or in parallel to Ngnr1. Further experiments show that SoxB1 genes are
direct target genes of Eya1 and Six1. Recent data suggest that Eya1 and Six1 play similar
roles in the central nervous system. First, overexpression of Eya1 and Six1 promote SoxB1-
positive proliferative progenitors as well as neuronal differentiation in the neural tube.
Second, Eya1 or Six1 knockdown strongly inhibits proliferation as well as neuronal
differentiation in the neural plate. The latter finding was unexpected, because both genes are
only weakly expressed in the neural plate. However, immunostaining with an Eya1-specific
antibody revealed stronger immunostaining in the neural plate/tube than in the pre-placodal
ectoderm suggesting that Eya1 protein (and possibly Six1 protein as well) is specifically
stabilized there. Taken together our results suggest that Eya1 and Six1 play essential roles
during two steps of both central and placodal neurogenesis: (1) they synergistically promote
the formation of proliferative neuronal progenitors and (2) they promote neuronal
differentiation in parallel to and/or downstream of Ngnr1.




Prohibitin: A novel neural crest specifier
Martina Schneider1, Doris Wedlich1, Alexandra Schambony2
1
  Zoological Institute II, Department of Molecular Developmental- and Cellphysiology,
University of Karlsruhe, Germany; 2 Biology Department, Developmental Biology, Friedrich-
Alexander-University Erlangen-Nuremberg, Germany

The prohibitins (PHB1 and PHB2) are remarkably conserved proteins present in multiple
cellular components of eukaryotic cells. In mammalian and yeast cells it is known, that the
PHB proteins form a complex at the inner membrane of mitochondria where they regulate
mitochondrial activities. Apart from this there are pleiotropic functions of prohibitin 1
described e.g. in cell cycle progression, apoptosis and regulation of adhesion and migration,
whereas a function in embryonic development is poorly examined.
Here, we report a novel role for prohibitin 1 regulating neural crest development in Xenopus
laevis. Xenopus prohibitin 1 (Xphb1) is expressed in the dorsal animal ectoderm at blastula
and gastrula stage. With neurulation it shows a highly specific expression pattern in the
neural plate and later in the neural folds, in the eye and in migrating neural crest cells. By
employing a loss of function approach using an antisense morpholino we could demonstrate
that XPhb1 represses the neural crest specifiers foxD3, slug and twist but does not alter the
expression of c-myc, snail and AP2. Depletion of c-Myc, however, resulted in loss of Xphb1.
To get XPhb1 into the hierarchy of neural crest specifiers epistatic studies based on c-Myc
function were performed. These experiments revealed that XPhb1 rescued the loss of slug
and twist expression caused by c-Myc knockdown. In sum our results show that XPhb1 acts
downstream of c-Myc, Snail and AP2 and upstream of FoxD3, Slug and Twist leading to a
modified view of neural crest induction including the novel component XPhb1.



                     Characterisation of FoxO genes in Xenopus laevis

Maximilian Schuff, Nabila Bardine, Doreen Siegel, Cornelia Donow and Walter Knöchel
Institute of Biochemistry, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

Forkhead box (Fox) transcription factors of subclass O are involved in important processes
like cell survival, proliferation, apoptosis, cell metabolism and the prevention of oxidative
stress. FoxO genes are highly conserved throughout evolution and their functions were
analysed in several vertebrate and invertebrate organisms. We have previously reported the
temporal and spatial expression patterns of FoxO1 and FoxO3 during embryogenesis of
Xenopus laevis. Our recent studies identified two new genes of this subclass, FoxO4 and
FoxO6. In situ hybridisation reveals a predominant expression of FoxO4 in the neural tube
and neural crest cells at tadpole stages. Later onwards, staining is visible in the brain and
eye, while FoxO6 only shows staining in the olfactory placode. The four FoxO genes show a
partial overlap of their spatial patterns, but by using quantitatative RT-PCR we found
significant differences in their expression behaviour during embryogenesis and in adult
tissues. We then tested all four FoxO genes by loss of function (LOF) and gain of function
(GOF) experiments. Injections of antisense morpholino oligonucleotides (MO) for FoxO1,
FoxO3, FoxO4 or FoxO6 resulted, in a dose dependent manner, in severe body reduction,
delay of development and, finally, in lethality. Interestingly, injection of FoxO4 MO leads to
defects in brain/eye formation and in heart development. Ectopic expression of FoxO1,
FoxO4, FoxO6 or C. elegans daf-16, which is the only FoxO gene in this species, leads to
failure in blastopore formation, while FoxO3 does not. These observations clearly indicate
that the various FoxO genes in Xenopus laevis, although being derived from a common
ancestor, have specialised for different functions.



Novel glogi gene 181 regulate protein trafficking during early Xenopus development

Ayako Sedohara1, Kaoru Sugimoto1, Akira Kurisaki1, Shinji Komazaki2, and Makoto
Asashima1, 3
1
  Department of Life Sciences (Biology), Graduate School of Art and Sciences, University of
Tokyo
2
  Department of Anatomy, Saitama Medical School
3
  International Cooperative Research Project (ICORP), Japan Science and Technology
Corporation (JST)


           We identified a novel golgi protein 181, containing four-transmenbrane domain and
highly conserved among vertebrate. 181 expressed ubiquitously until Xenopus late blastulae,
and localized in notochord at neurulae. Immunohistochemistry revealed that 181 colocalized
with golgi protein, suggesting that 181 exists in golgi apparatus. Overexpression of 181
induced gastrulation defect and upregurated the expression of Xbra (mesodermal marker),
Xnr1 and Chordin (organizer merker) at gastrulae. Loss of function of 181 also induced
gastrulation defect and modulated the expression of Xbra and Chordin severely at late
gastrulae. Electronmicroscopy showed that endoplasmic reticulum (ER) and golgi stack was
vesiculated in embryos disturbed the function of 181.

B56α and B56γ regulatory subunits of protein phophatase 2A inhibit both canonical
and noncanonical Wnt pathways in Xenopus laevis.
Sungmin Baek, Enobong Shammah, and Joni M. Seeling
Department of Biology, City University of New York, Queens College, Flushing, USA.

    Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase
2A, which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse
roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for
proteins with a conserved core domain and divergent amino- and carboxy-termini. The core
interacts with the highly conserved A and C subunits, while the variable domains provide
substrate specificity by binding diverse substrates. Ectopic B56α and B56γ reduce β-catenin
abundance and B56α reduces Wnt-dependent transcription, suggesting that B56α and B56γ
inhibit canonical Wnt signaling. In contrast, B56ε is required for canonical Wnt signaling. The
B56 gene family is highly conserved between mammals and Xenopus laevis. B56α, B56ε,
and two alternative splice forms of B56γ: B56δ/γ and B56γ/γ, are expressed in early Xenopus
development, but the precise roles of each of these isoforms in early Xenopus development
are not known. We have undertaken loss-of-function analyses of B56α, B56δ/γ, and B56γ/γ in
Xenopus laevis to shed light on their function during early Xenopus development. We found
that each of these B56 subunits play a role in noncanonical Wnt signaling. The microinjection
of anti-B56α, B56δ/γ, and B56γ/γ morpholino antisense oligonucleotides results in delayed
blastopore closure and convergent extension defects, as well as dorsalized embryos. Our
data suggest that the B56 family plays diverse roles in canonical and noncanonical Wnt
signaling during Xenopus development.


  Involvement of batrachotoxin binding sites in ginsenoside-mediated voltage-gated
                              Na+ channel regulation

  Tae-Joon Shin, Jun-Ho Lee, Byung-Hwan Lee, Sun-Hye Choi, Mi Kyung Pyo, Sung Hee
                      Hwang, Sang-Mok Lee, and Seung-Yeol Nah

 Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-
                                       701 Korea

Recently, we showed that the 20(S)-ginsenoside Rg3 (Rg3), an active ingredient of Panax
ginseng, inhibits rat brain NaV1.2 channel peak currents (INa). Batrachotoxin (BTX) is a
steroidal alkaloid neurotoxin and activates NaV channels through interacting with
transmembrane domain-I-segment 6 (IS6) of channels. Recent report shows that
ginsenoside inhibits BTX binding in rat brain membrane fractions. However, it needs to be
confirmed whether biochemical mechanism is relevant physiologically and which residues of
the BTX binding sites are important for ginsenoside regulations. Here, we demonstrate that
mutations of BTX binding sites such as N418K and L421K of rat brain NaV1.2 and L437K of
mouse skeletal muscle NaV1.4 channel reduce or abolish Rg3 inhibition of INa and attenuate
Rg3-mediated depolarizing shift of the activation voltage and use-dependent inhibition. These
results indicate that BTX binding sites play an important role in modifying Rg3-mediated Na+
channel properties.
             The establishment of cell polarity during Xenopus gastrulation
                   Asako Shindo, Takamasa S. Yamamoto, Naoto Ueno
                         National Institute for Basic Biology, Japan

 Cell polarity is an essential feature of animal cells contributing to morphogenesis. During
Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate
each other allowing anterior-posterior elongation of the embryo proper by Convergent
Extension (CE). Although it is known that the cellular protrusions at both ends of polarized
cells exert tractive force for intercalation and that Planar Cell Polarity (PCP) pathway is
known to be essential for the cell polarity, little is known about what triggers the cell
polarization and what the polarization causes to control intracellular events enabling the
intercalation that leads to the CE. We used EB3 (end-binding 3), a member of +TIPs that
bind to the plus end of microtubule, to visualize the intracellular polarity of chordamesoderm
cells during CE to investigate the trigger of the establishment of cell polarity. We found that
EB3 movement was polarized in chordamesoderm cells in relation to the notochord-somite
tissue boundary, and this polarity was observed independent of the change of cell
morphology.
 The polarized EB3 movement was also observed near the boundary between the
heterogeneous tissues induced by the distinct levels of nodal signaling or even during
contact with the non-physiological material. These results suggest that the polarity of the
chordamesoderm cells is coordinated along the mediolateral axis by sensing the tissue
separation. The role of the tissue interaction during cell polarization and the possibility as the
polarity cue will be discussed.



Induction of Apoptosis-Like Cell Dissociation and Body Axis-Bending in Polyamine-
Treated Xenopus Embryos and Rescue of High Temperature-Induced Apoptotic
Embryos by Exogenously-Microinjected Polyamines
Koichiro Shiokawa1, Takeshi Kondo1, Mai Aso1, Jun-Ichi Takai1, Shigeki Takubo1, Senji
Takahashi1, Masayuki Kajitani1, Yusuke Terui2, and Kazuei Igarashi2
1
  Depart. of Biosc., Sch. of Sci. and Engineer., Teikyo Univ., Utsunomiya 320-8551; 2Grad.
Sch. of Pharmaceut. Sci., Chiba University, Chiba 263-8522, Japan

Abstract: Exogenously-microinjected polyamines (putrescine, spermidine, and spermine)
either have no effects on development (at low concentrations) or stopped cleavage
immediately after microinjection (at high concentrations), and no other effects have so far
been reported. However, when these polyamines were simply added to the culture medium at
5-50 mM, Xenopus embryos undergo apoptotic cell dissociation at MBT. These polyamines
administered at lower concentrations (0.1 mM) did not induce the MBT-executed apoptosis,
but ventrally bend body axis of stage 40 tadpoles by interfering with arrangements of somites
and notochord in the ventral side. The stronger the basicity, the stronger their effects to
induce apoptosis and axis-bending. We also found that Xenopus embryos incubated at
relatively high temperatures (31-32 oC) undergo apoptosis-like cell dissociation at MBT.
Interestingly, fertilized eggs microinjected with polyamines, especially termine obtained from
thermophic bacteria, did not suffer from the heat-induced apoptosis-like developmental arrest,
and survived the high temperature (31-32 oC) to develop several hours longer. This suggests
that embryonic ribosomes formed from enormously-amplified rDNA might be unstable at
higher temperatures because of possible deficiency of protein components and polyamines
might help stabilize such ribosomes by binding to their RNA moiety.

The role of PTK7 in Xenopus neural crest migration
Iryna Shnitsar and Annette Borchers
Dept. of Developmental Biochemistry, GZMB, University of Goettingen, Germany
PTK7 is a transmembrane protein containing 7 immunoglobulin-like domains and a kinase
homology domain. Although the DFG triplet necessary for catalytic activity is mutated, the
overall structure of the kinase homology domain is conserved from Hydra to humans.
Knockout studies in the mouse revealed that PTK7 is required for neural tube closure and
establishment of inner ear hair cell polarity, processes that are regulated by PCP signaling.
Here, we demonstrate that Xenopus PTK7 is expressed in cranial neural crest cells and is
required for their migration. PTK7 loss-of-function leads to inhibition of neural crest migration
in whole embryos as well as in explanted neural crest cells. Furthermore, overexpression of
a deletion mutant lacking the kinase homology domain leads to a similar phenotype, like
PTK7 loss-of-function. Using the animal cap system we demonstrate that PTK7 recruits dsh
to the plasma membrane and that this function is dependent of the PDZ domain of dsh as
well as the kinase homology domain of PTK7. Moreover, overexpression of PTK7 leads to
phosphorylation of JNK indicating activation of PCP signaling. Currently, we are analyzing
the in vivo relevance of the PTK7-dsh interaction using combinations of loss- and gain-of-
function studies in whole embryos as well as neural crest specific expression of different
constructs using the Slug promoter.


The role of the RNA-binding protein Vg1RBP in Cranial Neural Crest (CNC) migration.

Michal Shoshkes and Joel K. Yisraeli
Department of anatomy and Cell Biology, Hebrew University - Hadassah medical school,
Jerusalem, ISRAEL.

The VICKZ family of RNA binding proteins is highly conserved in vertebrates and has been
implicated in many aspects of RNA regulation, including RNA stability, translational control,
and intracellular localization. The Xenopus homolog, Vg1 RBP/Vera, is expressed in oocytes,
where it binds several vegetally localized RNAs and co-localizes with them to the vegetal
cortex. In early embryos, Vg1 RBP mRNA is expressed throughout the neural plate and in
the sensorial layer of the epidermal ectoderm, and in later embryos is present in the
developing eye, otic vesicle and neural crest. By reducing Vg1 RBP expression via injection
of antisense morpholino
oligonucleotides (AMO) into 2-cell stage embryos, we observed that Vg1 RBP is required for
certain embryonic cell movements. Vg1 RBP AMO-treated embryos show inhibition of neural
crest and neural tube roof plate cells migration, although these cells initially express the
proper cell-specific markers. To probe whether Vg1 RBP is required in a cell autonomous
manner or not in CNC migration, I transplanted CNC from wild-type embryos into AMO-
injected embryos. Surprisingly, WT CNC showed no migration in AMO-injected embryos,
while CNC from AMO-injected embryos migrate in WT embryos. Injection of Vg1RBP-AMO
together with
fluorescent-labeled dextran into specific blastomeres in 32 cell-stage embryos also suggests
that Vg1 RBP is not required in CNC after exiting the tube.
In order to perform a detailed analysis of Vg1RBP protein expression during CNC
development, we examined the expression pattern in premigratory and migratory CNC in
chicks. Consistent with its apparent non-cell automous role during CNC migration, Vg1RBP
is not expressed in migratory CNC. We find, however, that Vg1RBP is expressed in
premigratory CNC, undergoing downregulation after exiting the neural tube. These results
suggest that Vg1RBP may play different roles at different stages in CNC development.



                   The role of Dppa-like in early Xenopus development
               Doreen Siegel, Maximilian Schuff, Ying Cao and Walter Knöchel
                     Institute of Biochemistry, University of Ulm, Germany
Pluripotency and self renewal in early embryos and ES cells require expression of distinct
genes, among them Oct-3/4 and Sox-2. While the existence of homologues of Oct-3/4 and
Sox-2 has been demonstrated in non-mammalian vertebrates, like Xenopus, the existence of
other early expressed genes, like developmental pluripotency associated genes (Dppa), has
not yet been documented. Dppa2 and its paralogue Dppa4 are found in mammalian ES cells
and early embryos, but their role in early development is still unclear. Based on protein
similarities to mouse and humans, we have identified a Dppa2/4-like gene (XDppa) in
Xenopus. The protein does not only contain a conserved SAP domain, but additionally a
conserved C-terminal region, that is also found in mammalian Dppa2/4. XDppa is maternally
transcribed, transcripts persist during early cleavage stages, but are severely diminished
after midblastula transition. In adults, we detected XDppa expression exclusively in ovaries.
In gain of function analyses, we observed similar phenotypes for XDppa and mouse Dppa2
or Dppa4 injected embryos. They all show formation of blastopore but suffer from defects in
blastopore closure and neural fold formation. Loss of function studies by using morpholino
antisense oligonucleotides reveal also no proper closure of the blastopore and the embryos
show even a total lack of neural fold formation and die before tailbud stage. To further
ascertain the relationship between the Xenopus and the mouse genes, we have tried to
rescue the XDppa loss of function phenotype by injection of mouse Dppa2 RNA. While
blastopore closure and neurulation are obviously rescued, death of embryos cannot be
prevented. Based upon this observation we conclude that mammalian Dppa2 can replace
XDppa during gastrulation and neurulation but that XDppa is required for additional functions.
This correlates to the findings that mammals contain at least 5 different Dppa genes whereas
only one is found in Xenopus so far.




Subcellular qPCR expression profiling in Xenopus laevis eggs

Radek Sindelka, Mikael Kubista

Abstract: Cell determination during early development directly depends on mRNA and
protein cell content and distribution. In mammalian cells, mRNA profiling is limited by small
amounts of RNA. In contrast, a /Xenopus/ egg contains very large amount of mRNA, which
opens for expression studies on the sub cell level. Here, we quantified mRNA levels of
several selected maternal genes in different sections of the /Xenopus/ egg. We determined
the amount of these mRNAs in egg sections along the animal-vegetal axis by real-time RT-
PCR. The experiments were performed on eggs before and after fertilization. Based on these
results a 3D map of key mRNAs in a single /Xenopus/ egg cell was constructed, that will give
clues to the relation between mRNA distribution and cell division and ultimately
differentiation.



Role of Gadd45 and DNA demethylation in Xenopus development

Lilian Sitter and Christof Niehrs
Division: Molecular Embryology, German Cancer Research Center, Heidelberg

DNA methylation is a key epigenetic modification that is essential for gene silencing and
genome stability in many organisms. Recently our laboratory discovered that Growth arrest
and DNA-damage-inducible protein 45 alpha (Gadd45α) a nuclear protein involved in
maintenance of genomic stability and DNA repair, plays a pivotal role in active DNA
demethylation. Gadd45α relieves epigenetic gene silencing by promoting DNA repair, which
erases methylation marks in Xenopus laevis oocytes and human cell lines. However, it
remains unknown if Gadd45 is involved in the regulation of DNA methylation in early
Xenopus development, where DNA demethylation is thought to precede zygotic transcription
of differentiation genes. MS-PCR on promoters of xbra, slug and myoD reveals site specific
DNA demethylation after midblastula transition. We are currently investigating if their
demethylation involves Gadd45. Gadd45 family proteins are differentially expressed during
Xenopus development. Overexpression as well as antisense morpholino mediated knock
down of XGadd45α or XGadd45γ cause pleiotropic developmental defects and retardation.
To identify Gadd45 regulated genes we performed a microarray analysis of Gadd45 depleted
gastrula and neurula stage embryos and are currently analysing the methylation dependent
regulation of these genes.


Cardiac morphogenesis defects caused by loss of the embryonic macrophage
secreted protein, LURP1.
Stuart J. Smith and Timothy J. Mohun. Email <ssmith@nimr.mrc.ac.uk>
Division of Developmental Biology, MRC National Institute for Medical Research, London,
UK.

The roles of macrophages during early embryonic development in vertebrates remain poorly
defined. They are known to phagocytose apoptotic cell corpses that arise during
development but may also act to deposit extra-cellular matrix components to specific
embryonic locations. In Xenopus, macrophages differentiate from anterior-ventral mesoderm
located adjacent to the heart-forming region around stage 20 and rapidly disperse throughout
the embryo along consistent migration routes. For example, initial movement of the rostral-
most macrophages from the ventral midline coincides with the onset of morphogenetic
changes that lead to heart tube formation. It is not known however if these macrophages
actively participate in cardiogenesis or whether their presence is merely coincidental.
Macrophages also localize to sites of blood vessel formation on the lateral flanks of the
tadpole but again, the significance is not known. A major challenge then is to impair
macrophage function and observe what affect this has on normal embryogenesis, especially
the forming cardiovascular system.

LURP1 is a small, secreted protein that belongs to the Ly6-superfamily whose target receptor
and function has not yet been determined. LURP1 mRNA is expressed exclusively in
differentiated embryonic macrophages, with no expression in the heart, blood vessels or their
cellular precursors. Morpholino-mediated inhibition of LURP1 expression in X. laevis and X.
tropicalis does not broadly interfere with macrophage migration. Nonetheless, it does cause
a critical failure in morphogenesis of the surrounding cardiogenic mesoderm. By stage 30,
the bilateral myocardial domains fail to elongate along the antero-posterior axis, fusion of the
myocardial plate on the ventral midline does not occur and no endocardial precursors can be
detected. Ultimately, the remnants of myocardium become buckled by force exerted by the
growing liver primordium. We conclude secreted LURP1 is necessary to permit correct cell
movements during tailbud stages of development and is supplied locally by the roving
macrophages.

xPitx3, a possible player in the Xenopus laevis somitogenesis process.
Smoczer C., Jerant L., Brode S., KoshrowShahian F., Wolanski M., Crawford M.J.
Department of Biological Sciences, University of Windsor, Ontario, Canada

Similar to other chordates, somitogenesis in Xenopus laevis starts at the neurula stage when
somite pairs bud off posteriorly along the dorsal axis in a rhythmic and progressive fashion.
Unlike other vertebrates, where somites undergo epithelialization prior to differentiating into
specialized tissues, amphibian somites are predominantly comprised of myotome and initiate
the myogenic program immediately after formation. There are different levels of regulation in
this intricate segmentation process, with multiple genes involved in the segmentation clock,
the somitogenesis wavefront, the cytoskeletal changes responsible for somitic cell behaviour,
and myogenic differentiation. Recent evidence suggests that Pitx3 is expressed in the
developed somites and all major muscle groups of the murine body wall and limb and that it
plays a role in the late differentiation of the myoblasts. [1] In our lab, we observed a similar
expression pattern in Xenopus embryos, while misexpression of this gene, by either
knockdown or augmentation, results in a bent dorsal axis due to the distortion of somite
structure and timing of development. This data, combined with microarray experiments
performed in our lab using xPitx3 knock-down embryos, identifies genes from all levels of
segmentation regulation as putative xPitx3 downstream targets, and has prompted us to
investigate the role of xPitx3 in this complex process.

1. L’Honore A et al. 2007 Developmental Biology 307(2): 421-433




         The role of IP3 signalling during embryonic wound healing in Xenopus

                                Ximena Soto and Enrique Amaya

The Healing Foundation Centre, Faculty of Life Science, University of Manchester,
Manchester M13 9PT, UK

        A major goal in regenerative medicine is to understand and ultimately facilitate our
body’s ability to repair itself following injury. As a first step toward this goal, we have begun to
investigate the molecular and cellular basis of embryonic wound healing, given that embryos
have the capacity to heal wounds quickly and completely. Tissue repair resembles embryo
morphogenesis in several ways, including cell migration, proliferation and differentiation. In a
screen for genes involved in morphogenesis in Xenopus, we identified an Ins(1,4,5)P3
phosphatase (IP3 phosphatase), which impairs embryonic wound healing, suggesting that
Ins(1,4,5)P3 signalling (IP3 signalling) plays a role in this process. This finding has motivated
us to investigate the role of IP3 signalling during embryonic wound healing, using Xenopus as
a model system. Two sets of enzymes regulate this pathway, the Ins(1,4,5)P3 3-kinases (IP3
kinases) and IP3 phosphatases. IP3 phosphatases, such as Ins(1,4,5)P3-5 phosphatase (IPP-
5), promote Ins(1,4)P2 (IP2) generation, which is an inactive product, while IP3 kinases
generate Ins(1,3,4,5)P4 (IP4), which is involved in calcium release modulation. We have
cloned IP3 kinase B and IPP-5 A from Xenopus tropicalis and have begun to investigate their
temporal and spatial patterns of expression using RT-PCR and in situ hybridization. In
addition, we are missexpressing these genes during early development in order determine
the effect of modulating IP3 levels in embryonic wound healing assays. Also, we are
localizing the expression of these genes in the epidermal region of later stages embryos in
order to determine the effect of modulating IP3 levels in epidermal wound healing. Finally we
are investigating the consequence of these manipulations on the organization of the
cytoskeleton such as analyzing microtubules, EB3 and tau2, or F-actin, moesin and
phalloidin-rhodamine. The aim of these studies will be to understand the role of IP3 signalling
in embryonic wound healing.


The RNA-binding protein XSeb4R plays a role in primary ectoderm formation in
Xenopus.
Jacob Souopgui, Jessica Vanhomwegen, Massimo Nichane and Eric J. Bellefroid
  Laboratoire d'Embryologie Moléculaire, Université Libre de Bruxelles, Institut de Biologie et
  de Médecine Moléculaires (IBMM), Gosselies, Belgium

  Maternal mARNs are key players of the first steps of development in vertebrates. Initially
  silenced in oocytes, these transcripts are translated after fertilization and play crucial roles in
  germ layer formation. We have previously shown that XSeb4R regulates endoderm and
  mesoderm formation by stabilizing and activating the translation of VegT mRNA. Here we
  provide evidence that XSeb4R might also play a role in the ectoderm by interacting with
  mRNAs encoding TGFβ/nodal antagonists such as Ectodermin and Foxi1E/Xema which are
  required for ectoderm specification. On the other hand, we also found that depletion of VegT
  in animal cap explants by use of morpholino oligonucleotides leads to a robust increase of
  ectodermal determinants. Together, these results suggest a model in which XSeb4R, by
  acting on both VegT and ectodermal determinants mRNA, plays an essential role in the gene
  homeostasis required for primary ectoderm formation.



A Temporal Modulation of FGF, Wnt, BMP and Retinoic Acid Signalling is Required for
Neural Crest Induction
Ben Steventon and Roberto Mayor
Department of Cell and Developmental Biology, University College London, London, U.K.

The neural crest is an embryonic stem cell population, whose induction has been shown to be
dependent of at least four different signalling pathways: BMPs, Wnts, FGFs and Retinoic Acid
signalling. However, whether these signals work at the same time or in a sequencial manner
remains to be analyzed. By asking when each signal is required for the induction of the neural
crest, the temporal problem of induction is addressed. Firstly, specific assays to separate three
distinct phases of the inductive process are established. Next, by using a combination of
inducible constructs and soluble proteins or inhibitor the role of BMP, Wnt, FGF and Retinoic
Acid signalling in each step is analysed. In the first phase, FGF signals are required along with
the inhibition of Wnt signals. Subsequently continued FGF signalling together with high Wnt and
intermediate BMP signalling is required. Finally, activation of all four pathways is required for
continued maintenance of the neural crest. Direct measurement of the activity of these
signalling pathways, by using reporter construct, supports a step-wise model of neural crest
induction. This work brings together previous work in the field and provides a single conserved
model for neural crest induction. In addition, a mechanism of induction is proposed in which the
sequence, duration and level of signalling activity is important in achieving a specific response.



   The Retinol Dehydrogenase-10 regulates retinoic acid signaling during axis formation
                        and patterning of the central nervous system
                                   Ina Strate, Edgar M. Pera
   Laboratory of Vertebrate Developmental Biology, Lund Strategic Research Center for Stem
             Cell Biology and Cell Therapy, Lund University, 22184 Lund, Sweden


  Retinoic acid (RA) is an important morphogen involved in many biological processes,
  including the development of the central nervous system (CNS). Its synthesis from Vitamin A
  (retinol) occurs in two steps, with the second reaction- catalyzed by retinal dehydrogenases
  (RALDHs) – long considered as most crucial for tissue-specific RA production in the embryo.
  We have recently identified the Xenopus homologue of retinol dehydrogenase-10 (XRDH10)
  that mediates the first step in RA synthesis from retinol to retinal. XRDH10 is specifically
  expressed in Spemann`s organizer and other domains of the early embryo that partially
  overlap with XRALDH2 expression. We show that endogenous RA suppresses XRDH10
gene expression suggesting negative feedback regulation. In mRNA-injected Xenopus
embryos, XRDH10 activated RA targets, including the organizer-specific Chordin gene , and
synergized with XRALDH2 in posteriorizing the developing brain. Knockdown of XRDH10 by
a specific antisense morpholino oligonucleotide had the opposite effects and caused
downregulation of RA responses and anteriorization of the brain. These data indicate that the
conversion of retinol into retinal is a developmentally controlled step and essential for axis
development and pattern formation of the CNS. We suggest that the nested gene expression
and concerted action of XRDH10 and XRALDH2 constitute a “biosynthetic enzyme code” for
the establishment of a morphogen gradient in the embryo.

Xenopus MIDLINEs control epithelial morphogenesis via microtubule organization
during neural tube closure
Makoto Suzuki, Naoto Ueno
Division of Morphogenesis, National Institute for Basic Biology, Okazaki 444-8585, Aichi,
Japan

To establish complex body plans, epithelial morphogenesis is one of the important
phenomena in which cell shape, motility and adhesiveness are regulated spatially and
temporally. The role of microtubule in such phenomena has been extensively evaluated by
using in vitro cell culture system, but its in vivo role is poorly understood. In searching for
developmental regulators that bind to microtubule, we found that MIDLINE1 (MID1) was
expressed in the neuroepithelium of Xenopus embryo. MID1 is a conserved microtubule-
associated protein and a responsive gene of Opitz G/BBB syndrome which is associated with
midline abnormalities such as hypertelorism, hypospadias and heart defects, suggesting
functions of MID1 in epithelial morphogenesis. In the present study, we analyzed the
developmental function of xMID1 and a paralogous gene (MID2). We show that EGFP-
tagged xMID1 highlighted filamentous structures and colocalized with microtubule in
embryonic cells. In addition, morpholino-induced knockdown of xMID1/2 disrupted neural
tube closure and induced neuroepithelial cell dissociation in higher-dose, suggesting
redundant functions of xMID1/2 in neural tube closure. To investigate the function of xMIDs
in detail, we performed Immunohistological and live-imaging analysis of morphants using
confocal microscopy. We found that neuroepithelial cells in morphants failed to elongate their
shape along apicobasal axis and constrict their apical side, and morphologies of these cells
became rounded rather than columnar. In addition, mediolateral intercalation of
neuroepithelial cells was also inhibited. Further analysis revealed that microtubule network
along apicobasal axis was disrupted in MO-injected neuroepithelial cells. These data suggest
that xMIDs function in neural tube closure via microtubule organization, which should be
required for epithelial cell shape, integrity and morphogenesis. We propose that similar
mechanisms underlie correct epithelial morphogenesis in other organs affected in Opitz
G/BBB syndrome patients.


“Don’t be so supPARficial: PAR-1 and neurogenesis”
Jacqui Tabler1, Olga Ossipova2, Hiroaki Yamanaka1, Jeremy Green1
1
  Deparment of Craniofacial Development, Kings College London.
2
  Department of Molecular, Cell and Developmental Biology, Mount Sinai school of medicine

During early development of Xenopus Laevis an epithelial monolayer is transformed into a
bilayer by cell divisions perpendicular to the plane of epithelium. These divisions produce a
superficial and deep layer of cells that are molecularly distinct. The deep layer will contribute
to primary neurogenesis and the superficial to secondary neurogenesis. The fate of these
two cell layers is defined by polarity cues such as atypical PKC (aPKC) and cell fate
determinants. PAR-1 has been shown to localise basolaterally in Xenopus ectoderm. Recent
studies show that PAR-1 may act in a novel way as a cell fate determinant that confers
primary neurogenesis within deep layer ectoderm and that its restriction to the basolateral
membrane is important for this function. This project investigates how PAR-1 is involved in
the specification of deep layer cells and hence primary neurogenesis. I present work that
suggests PAR-1 overexpression can cause trans-specification of superficial layer ectoderm
to deep layer fates. In addition I present fate analysis of these trans-specified primary neural
progenitors.


Crossveinless-2 Is a BMP Feedback Inhibitor that Binds Chordin/BMP to Regulate
Xenopus Embryonic Patterning
Vincent F. Taelman, Andrea L. Ambrosio, Hojoon X. Lee, Carrie Metzinger, Catherine
Coffinier and E.M. De Robertis
Howard Hughes Medical Institute and Department of Biological Chemistry, University of
California Los Angeles, CA 90095-1662, USA

Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize
BMP signaling. Through embryological and biochemical experiments we find that: 1) CV2
functions as a BMP4 feedback inhibitor in the ventral pole of the Xenopus embryo; 2) CV2
complexes with Twisted gastrulation and BMP4; 3) CV2 is not a substrate for tolloid
proteinases; 4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range);
5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid
digestion or to full-length Chordin/BMP complexes; 6) CV2 depletion causes the Xenopus
embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or of
tolloid inhibition. We propose that the CV2/Chordin interaction may provide a sink that
ensures a continuous flow of Chordin/BMP towards the ventral pole of the embryo. Tolloid,
which liberates BMP from Chordin inhibition, is required for the pro-BMP effects of CV2.

The role of xDel1 in A-P patterning of the neuroectoderm via non-BMP pathways
Akira Takai, Hidehiko Inomata, Mami Matsuo-Takasaki, Akiko Arakawa and Yoshiki Sasai
Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe,
Japan.

We previously reported that the secreted matrix molecule xDel1 functions as a regulatory
factor of D-V patterning in early Xenopus embryo. Attenuation of xDel1 function by
morpholino antisense oligos causes ventralization of the injected embryo as well as head
defects. Conversely, overexpression of xDel1 induces enlargement of dorso-anterior
structures. Reporter assays indicated that xDel1 attenuates BMP signaling by interfering with
the pathway downstream of the BMP receptor. Thus, xDel1 is an extracellular signaling
factor that controls D-V patterning by modifying BMP signaling during early vertebrate
embryogenesis. Interestingly, we found that xDel1 overexpression can induce head
structures in the secondary axis induced by Chd injection. This suggests that xDel1 can act
more than just regulating BMP signaling and affect directly antero-posterior (A-P) patterning
of the embryo.
In this study, we analyzed roles of xDel1 in A-P patterning in the neural ectoderm. We show
that attenuation of xDel1 in the presumptive neural ectoderm suppresses the expression of
anterior neural markers. Conversely, overexpression of xDel1 expands the expression of the
anterior markers and shifts mid-hindbrain markers posteriorly. Overexpression of anti-BMP
molecules did not cause these phenomena in the presumptive neural ectoderm, supporting
the idea that xDel1 also functions as a regulatory factor of head development via the control
of non-BMP signaling. We are currently analyzing physical and functional interactions
between xDel1 and patterning factors such as Wnt, Nodal and FGF, and will discuss the
results in our poster.


BMP signaling regulates pigment cell distribution in Newt embryo
Kazuhito Takeshima
Radioisotope Research Center, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

  The secreted growth factor BMP is a member of TGF-b super family, and plays various
important roles in vertebrate development including the distribution and patterning of pigment
cells. We studied this issue by using Japanese newt as a material. The pigment cells of the
newt become distributed throughout both the dorsal fin and the flank regions as stripes from
stage 36 onward. In this study, we analyzed the components of BMP signaling, i.e. ligand,
receptors, and antagonists, during the proliferation and the migration of melanophores.
  The artificial delivery of mRNA of these signaling components into the specific surface
regions of embryo was done by the electroporation method at stage 26-27 that no pigment
cell was detected in epidermis of Newt embryo. The incorporation of BMP4-mRNA into the
flank region caused a melanophore-free zone at the target site. GFP-mRNA was used as a
delivery and/or cell lineage marker in the all experiments. The electrical delivery of Noggin-
mRNA increased the number of melanophore and resulted in the expansion of the pigment
stripe. In some cases, melanophres spread out all over the abdominal region. We also
examined the constitutively active Alk3 and Alk2. The artificial delivery of these mRNAs
caused the similar reduction of melanophores as in the case of BMP. However, there was a
kind of difference in their regression pattern. The importance of BMP signaling in the
melanophore development and the mechanism of the pigment patterning in Newt embryo will
be discussed.

Extracellular regulation of FGF signaling in the early Xenopus embryo

Tan Hooi Min, Dobromir Iliev, and Edgar M. Pera

Laboratory of Vertebrate Developmental Biology, Lund Startegic Research Center for Stem
Cell Biology and Cell Therapy, Lund University, 22184 Lund, Sweden

Fibroblast growth factors (FGFs) play an important role in development and homeostasis,
and their activity needs to be precisely regulated to ensure proper signaling. We previously
presented the secreted serine protease inhibitor xHtrA1 as positive regulator of FGF
signaling in the extracellular space (Hou et al., 2007). The data suggested that xHtrA1
through cleaving proteoglycans release cell-surface bound FGF ligands and stimulate long-
range FGF signaling during establishment of the embryonic body plan (Gallagher, 2007). It is
obvious that, if not tightly controlled, the proteolytic activity of xHtrA1 would lead to an
unlimited amplification and propagation of FGF signals. We have isolated a full-length cDNA
clone encoding a secreted serine protease inhibitor (xSPI) that may act as a negative
regulator of xHtrA1/FGF signals. xSPI shows distinct expression in the early embryo and
promotes anterior development in mRNA-injected Xenopus embryos. xSPI mRNA induced
enlargement of head structures, suppression of mesoderm and reduction of neuronal
differentiation. These effects are reminiscent of those caused by knockdown of xHtrA1 or
inhibition of FGF signaling (Hou et al., 2007 and references therein). In contrast,
downregulation of xSPI by specific morpholino oligonucleotides caused microcephaly, a
phenotype that is also induced by misexpression of xHtrA1, FGFs or components of the
FGF-MAPK pathway. Moreover, xSPI mRNA prevented xHtrA1 from inducing ectopic tail-like
structures, mesoderm induction and stimulation of neuronal differentiation. Together, the
data suggest that xSPI may add another layer to the regulation of FGF signaling in the
extracellular space and via suppression of HtrA1 proteolytic activity restrict FGF activity in
the developing embryo.


References:
Hou S, Maccarana M., Min T. H., Strate I., Pera E.M. (2007). The Secreted Serine Protease
xHtrA1 Stimulates Long-Range FGF Signaling in the Early Xenopus Embryo. Developmental
Cell 13, 226-41.
Gallagher J (2007). Messages in the Matrix: Proteoglycans Go to the Distance.
Developmental Cell 13, 166-7.

     The PI3K signalling pathway is relevant for the directional migration of
Xenopus primordial germ cells
                                             Katsiaryna Tarbashevich and Tomas Pieler
            Dept. Developmetnal Biochemistry, GZMB, Justus-von-Liebig Weg, 11, 37077,
                                                                  Göttingen, Germany
                                                                    ktarbas@gwdg.de

        A number of novel PGC-specific mRNAs were identified in our lab by virtue of their
localization to the vegetal cortex and germ plasm in Xenopus oocytes. One of these
transcripts encodes for a novel Xenopus kinesin, xKIF13B. Morpholino-mediated knockdown
reduces average PGC numbers and results in severe PGC mislocalization. The rat
homologue of this particular kinesin was found to act in neurons as a motor protein directing
PIP3 coated vesicles to the region of neuronal outgrowth. In isolated germ cells, PIP3 is
enriched at membrane protrusions. This enrichment as well as the mode of protrusion
formation was disrupted in xKIF13B and PI3K knockdown cells. Inhibition of the PI3K
signalling in germ cells achieved by PGC-specific overexpression of a dominant-negative
version of PI3K, as well as of Xenopus PTEN, resulted in a similar phenotype as of xKIF13B
knockdown.
        Therefore, we propose that PI3K signalling and kinesin-dependent polarization of
Xenopus PGCs is relevant for the directional migration of these cells.

pMesogenin1 and 2 as target genes of Xtbx6 involved in Xenopus laevis
somitogenesis and myogenesis
Shunsuke Tazumi, Shigeharu Yabe, Jun Yokoyama, Yuko Aihara and Hideho Uchiyama
International Graduate School of Arts and Sciences, Yokohama City University, 22-2 Seto,
Kanazawa-ku, Yokohama 236-0027, Japan

T-box transcription factor tbx6 is expressed in posterior paraxial and lateral plate
mesoderms, and is involved in mesodermal differentiation. Basic-helix-loop-helix
transcription factor pMesogenin1 is required for paraxial mesodermal differentiation and
segmentation. To clarify the role of these genes in Xenopus laevis, we isolated
pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2
appeared to be transcriptional activators and were induced by Xtbx6-GR without secondary
protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential
T-box binding sites, and a reporter gene construct containing these sites was activated by
Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6
and pMesogenin1 and 2 knockdowns caused similar phenotypic responses, including somite
malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct
regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis,
including that of body wall muscle in Xenopus.

The synteny of the human X chromosome with the Xenopus tropicalis genome.
Radka Teichmanova, Jaroslav Macha, Tereza Tlapakova and Vladimir Krylov
Charles University in Prague, Faculty of Science, Department of Cell Biology, Czech
Republic.

Mammalian X and Y chromosomes have been shown to have arisen from an ancestral pair
of autosomes. The origin of sex chromosomes has been studied by comparisons of the
genomes of mammals, chicken and fish. Ancestors of most species of teleost underwent a
process of whole-genome duplication followed by losses of paralogous duplicates. This
process has complicated successive analyses. The recently-sequenced Xenopus tropicalis
genome and the creation of a genetic linkage map provide a new perspective on the
reconstruction of the X chromosome evolution. The study documented by this paper
compared the complete human X chromosome genetic map with data from chicken as well
as from X. tropicalis. Unmapped syntenic X. tropicalis scaffolds were localised into
chromosomes by TSA-FISH. The results show that nearly all genes of the human X
chromosome correspond to genes found in X. tropicalis chromosomes 2 and 10.



Ras-dva, a member of novel family of small GTPases, regulates the anterior ectoderm
  patterning during Xenopus laevis neurulation and functions in cells of secretory
                            organs in later development.
                 Tereshina Maria, Novoselov Vladimir, Zaraisky Andrey
         Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow

        Ras-like small GTPases are known to be involved in signalling cascades essential for
specification of the vertebrate body plan. Recently we have identified a novel Ras-related
small GTPase, named XRas-dva (dva – dorsal-ventral-anterior expression in Xenopus
midneurula stage embryo). We established that XRas-dva and its homologues constitute
novel family of small GTPases. To understand their specific functions we investigated the
XRas-dva expression pattern and its regulation, effects caused by XRas-dva down-regulation
and protein intracellular localization. The expression of XRas-dva starts during gastrulation in
the anterior ectoderm being directly up-regulated by the homeodomain factor Otx2. However,
by the midneurula stage due to inhibitory influence of another homeodomain factor - Xanf1,
the XRas-dva expression is confined to the crescent-shaped area surrounding the anterior
neural plate and corresponding to the pre-placodal ectoderm, cement gland and neural crest.
The experimentally provoked down-regulation of XRas-dva elicits inhibition of several early
expressing markers of the anterior neural plate and neural folds. In contrast the epidermal
regulator BMP-4 and its target genes are up-regulated. By the tadpole stage, this results in
severe malformations of the forebrain, head cartilages and sense organs. Basing on these
results and on the data indicating intracellular membrane localization of XRas-dva, we
proposed its involvement in regulation of FGF signalling in early development. In support of
this we showed that during neurulation XRas-dva can interfere with FGF8a signalling in cells
of anterior margin of the neural plate. Together, the data obtained indicate that Ras-dva is an
essential component of signalling network that patterns the early anterior neural plate and
the adjacent ectoderm in Xenopus embryos. Besides we established that on late stages
XRas-dva is specifically expressed in the cells of secretory organs (cement gland, hatching
gland, oesophagus, stomach etc.). Such a correlation may indicate possible involvement of
XRas-dva in secretory processes in differentiated cells.




TEF-1 expression and function during early development
François Naye, Corinne Faucheux, Sandrine Fédou, Nadine Thézé and Pierre Thiébaud.
UMR CNRS 5164 Université Victor Ségalen Bordeaux 2. 146, rue Léo Saignat
33076 Bordeaux Cedex, France

We have cloned and analyzed the expression of two Xenopus laevis genes encoding TEF-1
proteins that are members of the conserved family of transcription factors containing a TEAD
domain. The two genes are maternally expressed and with a dynamic pattern in early
embryo where the major domains of expression are in the brain, eye and somites. In animal
cap assay, the two genes are activated by bFGF but are differently regulated by BMP4 and
the muscle regulatory factor Mef2d.
We have used Engrailed repressor (EnR) and VP16 activator fusion constructs to analyze
the function of TEF-1 in the embryo. Moreover a morpholino base approach was used to
knock down the two genes in the embryo. The resulting phenotypes obtained with those
approaches indicate an important function of TEF-1 genes in the early patterning of the
embryo and more specifically in the BMP signaling pathway.




Investigating the origin of long-term dividing neural progenitors during Xenopus
development
Raphael Thuret, Sarah Hodge, Akiko Tsutsui and Nancy Papalopulu
M. Smith Building, Faculty of Life Science, University of Manchester, Oxford Road, M13 9PT,
Manchester

        It is thought that the development of Xenopus central nervous system (CNS) is
divided in two periods, primary neurogenesis during neurulation and secondary neurogenesis
during metamorphosis, allowing the formation of adult CNS. At early neurula stage, the
Xenopus neural plate is composed of a bilayered epithelium and most of the primary neurons
originate from the deep layer of this epithelium whereas cells of the superficial layer remain
as progenitors.
        The aim of our work is test the hypothesis that the superficial layer progenitors are
preferentially dedicated to secondary neurogenesis. To characterise secondary
neurogenesis better, overall proliferation in CNS has been followed from NF33/34 to NF66 by
PH3 immunostaining with the idea that the second wave of neurogenesis will be preceded by
a burst in progenitors proliferation. A clear increase in proliferation has been identified at
NF56, during metamorphosis. In order to determine the contribution of superficial and deep
layers cells to differentiation, we performed homotopic grafts of labelled superficial and deep
cell layers and assessed their differentiation between NF38 and 42. We report that superficial
grafts start to differentiate already at NF38 but a big proportion of these cells keep a
progenitor identity. Inversely, most of the deep layer grafted cells are already differentiated at
all stages analysed. Our results so far do not show a black-and-white difference between
deep and superficial layer cells, as we had imagined. Nevertheless a quantitative difference
in the amount of differentiation derived from deep or superficial layers is observed. By
carrying the analysis to later stages, we will see whether the deep layer progenitors are
depleted earlier than the superficial ones.


Xenopus tropicalis pre-Sertoli cells support growth of germinal stem cells with
cocktail of growth factors.
Tereza Tlapakova1, Jaroslav Macha1, Vladimir Krylov1, Jana Sevcikova Jonakova1, Natasa
Sebkova1, Monika Sidova1 and Paul Richardson2
1
  Charles University in Prague, Faculty of Science, Department of Cell Biology, Czech
Republic. 2Department of Energy Joint Genome Institute, USA.

A culture of pre-Sertoli cells was prepared from testis of juvenile X. tropicalis. Cell type
identification was based on the expression of Sertoli cell markers, namely Sox9, WT1,
NCAM, SF1 and transferrin. The cell growth enhanced by conditioned medium reflects the
presence of autocrine growth factors. Moreover, pre-Sertoli cells support growth of colonies
of germinal stem cells. Genome-wide expression screening of pre-Sertoli cells identified 34
growth and differential factors with intermediate or high expression. The results were verified
by RT-PCR. Comparison of pre-Sertoli cell and testis expression profiles revealed
approximately 200 genes with 100x drop of expression in an adult organ. These genes may
be involved in Sertoli cell differentiation.



Expression pattern of Ric-8 during Xenopus embryo development.
Maldonado R., Hinrich M.V., Olate J and Torrejón M. Department of Biochemistry and
Molecular Biology. Biological Sciences Faculty. University of Concepción. Email:
matorrejon@udec.cl.

Asymmetric cell division is an essential process to control cell differentiation during
embryogenesis in order to generate the different type of cells in an adult organism. Recently
we have cloned a gene from Xenopus embryos coding Ric-8 which has a GEF (Guanine
Exchange Factor) activity for Gas subunit. Studies in C. elegans and Drosophila have shown
that Ric-8 plays an important role during the asymmetric cell division controlling the correct
mitotic spindle alignment during early steps of embryogenesis. In our laboratory, we have
started to study the expression of this gene during different stages of Xenopus
embryogenesis, and we have shown that Ric-8 plays an essential role during early stages.
The results show the presence of Ric-8 mRNA from non-fertilized egg until embryo stage 42
with a strong expression at neural level, somites and craniofacial. Also, loss of function
analysis at early stages of embryogenesis shows that Ric-8 is essential for a correct cell
division. Finally we have generated a transgenic animal in X. tropicalis with the green
fluorescent protein (GFP) as a reporter gene under the human isoform Ric-8B promoter to
follow the expression patter and study the regulation of this gene in vivo. These results
support studies from other investigators performed in mouse and indicate that Ric-8 plays an
essential role during early development in X. tropicalis especially at neural system level.

The Role of Bicaudal-C in Kidney Development of Xenopus
U. Tran1, L. Zakin2, A. Schweickert3, R. Döğer1, M. Blum3, E. M. De Robertis2 and O.
Wessely1
1
  LSUHSC, New Orleans, LA, USA
2
  HHMI, UCLA, Los Angeles, CA, USA
3
  University of Hohenheim, Stuttgart, Germany

    The RNA-binding molecule Bicaudal-C (BicC1) has been shown to regulate embryonic
development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not
show early developmental defects, but develop polycystic kidney disease (PKD) instead.
PKD is among the most common single gene diseases in humans, is the leading cause of
end-stage renal failure and requires extensive treatments, such as dialysis and kidney
transplantation. It is characterized by the uncontrolled proliferation of kidney epithelial cells
and defects in transepithelial solute transport resulting in the formation of fluid-filled cysts.
    To further investigate the molecular mechanism of Bicaudal-C in kidney development, we
analyzed its function in the developing Xenopus pronephros. Inhibition of the translation of
endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) resulted in a
“PKD-like” phenotype. Embryos developed generalized edemas and dilated pronephric
tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros
exemplified by the loss of molecular markers expressed in the late distal tubule. Mechanistic
analyses suggest that BicC1 functions by regulating the expression of Polycystin-2 (PC2).
BicC1 mutant mice exhibited reduced expression of PC2 protein and RNA in the kidney.
Moreover, depletion of endogenous PC2 protein in Xenopus by antisense morpholino
oligomer knockdown resulted in a “PKD-like” phenotype highly similar to the one observed in
Xenopus embryos lacking BicC1. Moreover, molecular analyses showed that BicC1 and PC2
cooperate and PC2 is required for the activity of BicC1. Thus, we propose that the PKD
phenotype by BicC1 is caused - at least partially - by misregulation of PC2.
Caracterization and role of C3H protein during Xenopus laevis embryonic
development
Karine TREGUER, Corinne Faucheux, Sandrine Fédou, Pierre Thiébaud and Nadine Thézé.
UMR CNRS 5164 Université Victor Ségalen Bordeaux 2. 146, rue Léo Saignat
33076 Bordeaux Cedex, France

C3H proteins are RNA binding proteins witch control RNA stability. These proteins contain
two tandem zinc-finger domains, each with a cystein histidine repeat in a cys cys cys his
configuration (C3H), they are encoded by a multigenic family gene. This family is composed
of three genes in mammalians and four in amphibians and fishes.
During my thesis one part of my work is to characterise the expression pattern and the
function of the four C3H genes during xenopus development. First of all, I have established
the spatio-temporal pattern of expression of the four genes by BT-PCR and in situ
hybridization. In a second part, I have studied the function of these genes using a gain and a
loss of function strategy. My results show that the C3H proteins have an important role
during embryonic development, during the patterning of the somites and the pronephros
differentiation.

The homeobox gene Goosecoid acts as a regulator of Planar Cell Polarity (PCP)
Bärbel Ulmer, Philipp Andre, Kirsten Deißler, Verena Mauch, Axel Schweickert and Martin
Blum
University of Hohenheim, Institute of Zoology, Stuttgart, Germany

Goosecoid (Gsc) was the first organizer-specific gene cloned from Xenopus. Ventral
injections induced secondary axis formation with high frequency. The Gsc knockout mouse,
however, surprisingly revealed no gastrulation defects. To study Gsc in the mouse by gain-
of-function, Gsc was conditionally misexpressed under the control of the Brachyury streak
enhancer. Mid-gestation embryos displayed neural tube closure defects (NTD) with about 20
% penetrance upon mild misexpression. Massive misexpression resulted in embryonic
lethality at mid-gastrula stages. Notochord formation and dorsal extension were
compromized in affected embryos. Specificity was demonstrated by massive NTD in
chimaeric mouse embryos generated with ES cells which massiveley overexpressed Gsc. In
order to investigate whether this result was a mouse-specific effect, we misexpressed Gsc
protein from an inducible Gsc-GR construct in Xenopus. Dorsal injections induced NTDs at
very high frequency when Gsc was activated between stage 6 and 11, i.e. before and during
gastrulation. Marker gene analyses showed, that NTDs were not due to patterning defects.
As NTDs were reminiscent of phenotypes induced upon interference with the PCP-Pathway,
we hypothesized that Gsc might act as a transcriptional regulator of PCP. Therefore,
downstream components of PCP were assayed for their potential to rescue Gsc-induced
NTD. Indeed, the three components analyzed so far, constitutively active RhoA, strabism and
prickle all were able to partially revert Gsc phenotypes with high statistical significance.
These unexpected findings might help to explain the lack of a gastrulation phenotype in
knockout mice. In addition, we like to suggest a role for the endogenous Gsc gene in
prechordal mesodermal cells, i.e. in switching from convergent extension (notochord) to
migration (prechordal mesoderm).

The X. tropicalis mutation kaleidoscope truncates the copper transporter ATP7a and
models Menkes Disease
Elisenda Vendrell and Lyle Zimmerman

The chemically-induced X. tropicalis mutation kaleidoscope (kal)1, results in strikingly variegated
retinal pigmented epithelium (RPE), and is among the first mutations in X. tropicalis to be genetically
mapped. Kal lens and retinal neuroepithial layers are properly organized, but gaps are observed in
the RPE layer. Mutants also show reduction in melanocyte number as well as specific cartilage
defects. We used a meiotic mapping approach to define a genetic interval containing the kal
mutation between two simple sequence repeat polymorphisms in Scaffold 10 (v4.1) 0.5 Mb apart on
X. tropicalis Linkage Group 5 2. The kal interval includes the ATP7a copper transporter, defects in
which cause Menkes disease, whose symptoms include pigmentation and connective tissue
abnormalities reminiscent of kal. Analysis of the kal ATP7a locus revealed a base change at the
exon 20 splice acceptor, resulting in mis-spliced mRNA deleting a domain responsible for
subcellular localization of ATP7a to the trans-golgi network. Kaleidoscope demonstrates the utility of
X. tropicalis genetics in developing models for human disease, and may be a substrate for small
molecule or gain-of-function suppressor/enhancer screens to help develop therapies for Menkes
Disease.
1
    Goda, T. et al. Plos Genetics (2006) Vol 2: 0811-0825.
2
    Sater & Wells (www.tropmap.biology.uh.edu)


Knockdown of PKD2 results in left-right axis defects in Xenopus laevis
Philipp Vick, Axel Schweickert, Susanne Bogusch, Tina Beyer, Thomas Weber and Martin
Blum
University of Hohenheim, Institute of Zoology, Stuttgart, Germany

In all vertebrate species the normal asymmetric arrangement of the inner organs was shown
to be under control of the highly conserved left-sided Nodal signaling cascade. Furthermore,
in mammals, fish and - recently - amphibians, a cilia-driven extracellular fluid flow was
demonstrated to be required for symmetry breakage in early neurula embryos, immediately
prior to asymmetric gene expression. However the mechanism how the laterality defining
signal gets translated into asymmetric gene activity in the left lateral plate mesoderm is not
understood to date. In mice knockout of the polycystic kidney disease gene 2 (PKD2), which
encodes a ciliary ion channel, results in cystic kidneys and left-right defects. This is thought
to be caused by the absence of a left-sided calcium signal, normally generated via PKD2 in
response to flow. We cloned the Xenopus homolog of PKD2, analyzed its mRNA and protein
expression during early development, and performed morpholino-mediated knockdown
experiments. PKD2 mRNA was maternally expressed throughout the animal hemisphere of
the embryo. Zygotic transcripts were found on the dorsal lip, in the neuroectoderm and the
forming kidney. Polycystin-2 protein localized to motile monocilia of the gastrocoel roof plate
(GRP), i.e. the tissue which generates flow. PKD2 gene knockdown resulted in loss of left-
sided marker gene expression and cystic embryos. Surprisingly, flow was absent in
morphants. Scanning electron microscopy revealed altered morphology of GRP tissue,
namely an apparent fate change of GRP cells to that of neigboring endodermal cells. This
notion was further supported by marker gene analysis. Together these data suggest a role
for PKD2/calcium in the morphogenesis of the GRP in the frog.

Expression profiling of inner ear, brain, and kidney transcriptomes reveals candidate
genes for differential gene expression

Selene M. Virk , TuShun R. Powers, Casilda Trujillo-Provencio, and Elba E. Serrano
New Mexico State University, Las Cruces NM 88003 USA

The emerging emphasis on integration of information gathered at the cellular level into a
system level understanding of health and disease has heightened interest in the molecular
underpinnings of organ function. Here we present results of comparative microarray studies
that were undertaken with the goal of determining whether unique genes or gene networks
are differentially expressed between Xenopus inner ear, brain, and kidney. These organs
were chosen because of our interest in the inner ear and neural regeneration, and because
of the pharmacological similarities between the inner ear and kidney. We used the
Affymetrix GeneChip® Xenopus laevis Genome Array to examine the transcriptomes of
brain, kidney, and inner ear organs from juvenile X. laevis. Although the Affymetrix
microarray contains 15,491 non-control probe sets, the degree of transcript annotation for X.
laevis is limited, and many probe sets have no gene designation. We therefore focused this
initial study on the 61% of probe sets that were associated with an Entrez gene ID. Brain,
kidney, and inner ear microarray data were normalized using the RMA module of the Affy R
package, then analyzed with the Maanova R statistics package (www.bioconductor.org).
Differentially expressed genes were indentified in pairwise comparisons of organ expression
data. When a False Discovery Rate of <= 0.01 and fold change of at least ±2 were used as
the criteria for differential expression, 1965 (21%) probe sets emerged as candidate
regulatory genes that are potentially specific to organ function. Of these 1965 probe sets,
44.6% were unique to a single comparison, 49.5% were present in two comparisons, and
5.9% were common to all comparisons. Due to gaps in annotation of the X. laevis
transcriptome, our future challenge is to extract functional meaning from these patterns of
differential expression. Research Support: NSF (IGERT-DGE-0504304), NIH (GM008136;
DC003292; P50GM068762).


p36RITA: A novel RBP-Jk interacting protein affects neuronal differentiation
Stephan A. Wacker1, Franz X. Oswald2, Cristobal Alvarado2, and Walter Knöchel1
1
  Institute of Biochemistry, 2Institute of Internal Medicine I, University of Ulm, 89081 Ulm,
Germany

Notch proteins are the receptors for a highly conserved signaling pathway that regulates
numerous cell fate decisions during development. This signal transduction involves the
presenilin-dependent intracellular processing of Notch, and nuclear translocation of the
intracellular domain, Notch-IC. Notch-IC associates with the DNA binding protein RBP-
Jκ/CBF-1 to activate transcription of Notch target genes. In the absence of Notch signaling,
RBP-Jκ/CBF-1 acts as a transcriptional repressor through the recruitment of histone
deacetylase (HDAC) corepressor complexes.
We identified p36RITA as a novel RBP-Jκ/CBF-1 interacting protein in a yeast two-hybrid
screen. p36RITA consists of 269 amino acids and does not show significant homologies to
other proteins. Using GFP-fusion proteins, we found that p36RITA associates with tubulin in
the cytoplasm. Interestingly, after the inhibition of nuclear export by leptomycine B, p36RITA
accumulated in the nucleus. By using deletion constructs, we identified a tubulin interaction
domain at the very carboxy-terminus and an amino-terminal domain necessary for nuclear
export. RBP-Jκ interacts with the middle part of the protein sequence. Therefore, we named
this novel 36 kDa protein p36RITA (RBP-Jκ Interacting and Tubulin Associated). In Xenopus
laevis embryos, a knockdown of xRITA resulted in a dramatic loss of primary neurons,
comparable to the effect of the overexpression of a dominant active Notch-IC. At later
stages, the knockdown of xRITA resulted in developmental defects of the eyes, head
structures and the heart. We propose a model in which regulated nucleocytoplasmic shuttling
of RBP-Jκ together with p36RITA may be a novel regulatory mechanism in the Notch
signalling pathway.


Recombinase and integrase mediated gene interference in Xenopus development

Christoph Waldner, Kathrin Sauert, Magdalena Roose, Gerhart U. Ryffel.
Institut für Zellbiologie (Tumorforschung), Universität Duisburg-Essen, Hufelandstr. 55,
D-45122 Essen, Germany

Expression of transgenes in Xenopus offers several advantages compared to injecting
mRNA encoding a given protein of interest: The expression pattern obtained in transgenic
strains is more reproducible and can be directed to specific tissues. Furthermore, the
expression is not limited to the early stages of development. However, investigating factors
that interfere with development an inducible system is essential. We established the Cre/loxP
binary system in Xenopus laevis and generated permanent transgenic Cre recombinase
strains. To obtain temporal controlled Cre expression we established the HSP70:Cre strain
HSPCre1 inducible by heat shock that can induce a reporter gene at will. We used this strain
to activate a dominant negative mutant of the transcription factor HNF1b, which is a known
regulator of kidney development. After induction by heat shock at gastrula stage, mutated
HNF1b leads to decreased pronephros size and massive edemas at the larval stage
indicative for kidney dysfunction. We thus conclude that the heat shock inducible HSPCre1
strain is a valuable tool to dissect the temporal function of regulators in Xenopus
development.
Another enzyme that can be used for genetic manipulation in Xenopus is the FC31 integrase
that recombines the specific recognition sites attP and attB resulting in a unidirectional
integration event of an attB site bearing plasmid into an engineered attP site. We have
generated transgenic strains with a stably integrated attP site fused to a ECFP marker gene
(docking site). Preliminary experiments show that site specific integration into this docking
site is in principle possible by co-injecting FC31 mRNA and an attB site bearing integration
plasmid into one-cell staged F1 embryos of such a docking site strain. This technique has the
potential to allow site specific transgenesis in Xenopus, because one copy of any gene-of-
interest can be integrated into a predetermined locus.

Gastric H+/K+ATPase functions in cilia-driven Leftward Flow in Xenopus left-right
development
Peter Walentek, Tina Beyer, Thomas Weber, Axel Schweickert and Martin Blum
University of Hohenheim, Stuttgart, Germany

Establishment of the left-right (LR) body axis is essential for the correct positioning of
asymmetric organs, e.g. heart, digestive system and pulmonary lobes, during vertebrate
embryogenesis. The gastric ion pump H+/K+ATPase (subunit alpha) was initially described to
be asymmetrically expressed during early cleavage stages of X. laevis development. Based
on this finding the so-called "ion-flux hypothesis" was put forward, which postulated that early
asymmetries in ion distribution accounted for symmetry breakage during cleavage. The
recent demonstration of cilia-driven Leftward Flow in Xenopus LR axis specification raised
the question if and how early H+/K+ATPase asymmetries are connected to flow. We therefore
re-investigated early expression patterns of H+/K+ATPase. No consistent asymmetries could
be detected. H+/K+ATPase subunit alpha mRNA was found in the animal hemisphere
throughout the cytoplasma from early cleavage up to blastula stages. Analysis of protein
expression by immunohistochemistry revealed localization of both alpha and beta subunits
on cilia of gastrocoel-roof plate (GRP) cells, i.e. the transient cilia-bearing epithelium which
generates Leftward Flow on the dorsal side of the archenteron in the neurula embryo.
Treatment of embryos from stage 9.5 onwards with the drug SCH28080, a specific inhibitor
of H+/K+ATPase function, resulted in LR defects, strongly suggesting a function of
H+/K+ATPase from gastrulation onwards. Gene knockdown targeted to the GRP confirmed
an involvement of both subunits in LR development. Flow was severely affected in
morphants, implementing H+/K+ATPase in Leftward Flow without a prior asymmetric function
during earlier embryogenesis. In summary our results are consistent with Leftward Flow as
the singular event of symmetry breakage in Xenopus.

The parkin-co-regulated gene (PACRG) - a highly conserved cilia-marker in Xenopus
left-right development
Thomas Weber, Nina Tietze and Martin Blum
University of Hohenheim, Institute of Zoology, Stuttgart, Germany

PACRG was first described as a gene co-regulated with parkin/Park2, a Parkinson's disease
related gene. Both genes share a bi-directional promoter. Besides a role in Parkinson’s
disease and leprosy, PACRG has been involved in the ubiquitin-proteasome system. In
addition, PACRG has been described as a protein of yet unknown function located along the
entire axoneme of cilia and flagella. As an entry point into dissecting the ciliary role of
PACRG we started to analyze the gene in its function for left-right (LR) development. A
comparative expression and sequence analysis in rabbit, mouse and frog embryos revealed
high conservation of amino acid sequences. Different isoforms resulting from alternative
splicing were detected in frog, mouse and rabbit. A full-length PACRG cDNA comprising all 6
exons, however, was not found. Analysis of PACRG gene expression revealed conserved
expression domains during embryogenesis. During gastrula/neurula stages, PACRG marked
the monociliated epithelium at which cilia-driven leftward flow breaks the bilateral symmetry
of the embryo, i.e. the posterior notochord (PNC) in mouse/rabbits and the corresponding
structure in Xenopus, the gastrocoel roof plate (GRP). At later embryonic stages, PACRG
was found in every ciliated cell. In order to functionally assess PACRG on LR cilia, loss of
function experiments in Xenopus laevis neurulae using antisense morpholinos were
preformed. Dose-dependently, altered marker gene expression was detected. The conserved
embryonic PACRG expression pattern and loss of function experiments in Xenopus suggest
the involvement of PACRG in leftward flow. In summary this work involves PACRG as a pan-
ciliary marker in the development of vertebrate laterality.

An improved genetic map for Xenopus tropicalis.
Dan E. Wells1, Zhenkang Xu1, Laura Gutierrez1, Vladimir Krylov2, Matthew Hitchens3, Tereza
Tlapakova2, Renata Slavikova2, Peter Blythe3, Yuan Ye3, Jaroslav Macha2, Steven E.
Scherer3, and Amy K. Sater1.

1 Department of Biology and Biochemistry, University of Houston, Houston TX USA
2 Department of Cell Biology, Faculty of Science, Charles University in Prague, Czech
Republic
3 Human Genome Sequencing Center, Baylor College of Medicine, Houston TX USA

We have used a bioinformatics-based strategy to identify unique SSLPs within the Xenopus
tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for
SSRs using a modification of Tandem Repeat Finder; unique SSRs were then tested for
amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast
individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of
DNA samples from 192 F2 individuals. Based on these genotypes, linkage analysis was
carried out using JoinMap 3.0. Over 3000 SSLPs were genotyped, yielding a genetic map of
over 2900 SSLP markers, consisting of 10 linkage groups, and 3 smaller clusters. Linkage
groups and clusters were assigned to X. tropicalis chromosomes through the physical
mapping of cDNA probes derived from genes included in respective scaffolds. For this
purpose, fluorescence in situ hybridization coupled with a tyramide amplification step (FISH-
TSA) was performed. Each LG and cluster was mapped to individual chromosomes with no
duplication. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI)
indicate that the map provides minimal representation of 888 MB, comprising over 52% of
the X. tropicalis genome. The genetic map and SSLP marker database constitute an
essential resource for genetic and genomic analyses in X. tropicalis.


    A chemical genomic screen in Xenopus identifies small molecules affecting cell
                                      migration
 1                      2
   Matthew L. Tomlinson, Richard M. White, 2Leonard I. Zon, 3Robert A. Field and 1Grant N.
                                            Wheeler.
      1
       School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.
       3
         Dept. of Biological Chemistry, The John Innes Centre, Norwich, NR4 7UH, UK.
                 2
                   HHMI, Children's Hospital of Boston, Boston, MA 02115, USA

      Chemical genomics is a powerful tool for identifying novel small molecules. The premise
of chemical genomics is that, with the aid of appropriate high throughput synthesis (chemical
and/or enzymatic) and biological screening strategies, it may be feasible to identify specific
small molecule inhibitors of any biochemical event. Such phenotype based hit-to-lead
screens with either cell-based assays or model organisms have in recent years uncovered a
number of tools for developmental biology investigations.
      We have carried out a forward chemical genomic screen to identify modulators of
melanophore (pigment cell) development in Xenopus laevis. Pigment cells because of their
ease of visualization provide an ideal system to identify compounds affecting cell
differentiation, morphology and migration. The results of the screen will be presented.
      Two compounds identified in the screen affecting pigment cell migration are NCS 84093
and MLT-013. NCS 84093 specifically affects pigment cell migration in the developing
embryo and we have determined it to be a novel matrix metalloproteinase (MMP) inhibitor. In
addition to their function in cell migration MMPs have important roles in inflammation,
angiogenesis and tumour metastases.
      Compound MLT-013 has a stronger effect than NCS 84093 and completely inhibits
melanophore cell migration through an as yet undetermined mechanism. An advantage of
chemical genomics is that compounds can easily be tested in other systems. When tested in
a zebrafish melanoma model MLT-013 was found to reduce melanoma formation.
      Thus, through a combination of chemical genomics and developmental biology we have
identified a novel class of MMP inhibitor and a compound with potential anti-cancer
properties.


The Gsx Transcription Factors In Xenopus Tropicalis: Regulating Primary
Neurogenesis
E.F. Winterbottom1, J.C. Illes2, H.V. Isaacs1
1
  University of York, UK
2
  University of Sheffield, UK

The Gsx proteins are members of the ParaHox family of homeodomain transcription factors.
We have cloned two Gsx genes, Gsh1 and Gsh2, from the frog Xenopus tropicalis, and
shown that they have complex and dynamic patterns of expression in the developing CNS.
Of particular interest is the expression of Gsh2 during primary neurogenesis in the
intermediate column of the open neural plate, where primary interneurons form.
Interestingly, the Drosophila Gsx homologue, ind, is expressed in a corresponding
intermediate domain of the embryonic neurectoderm, and is essential for the specification of
neurons arising from that domain. This suggests a potentially conserved role for Gsx factors
in neural regionalization throughout metazoans. We have used antisense morpholinos and
domain fusion constructs to investigate the function of Gsh proteins in Xenopus primary
neurogenesis. Overexpression and inhibition experiments indicate that Gsh function is
required for the normal development of primary interneurons. Furthermore, our data indicate
that Gsh proteins are able to up-regulate BMP signaling in the embryo, and support a model
in which they act as transcriptional repressors to modulate the expression of Iroquois neural
pre-pattern genes at open neural plate stages.


Myosin-10 and actin filaments are essential for mitotic spindle function
Sarah Woolner1#, Lori O’Brien2, Christiane Wiese2 and Bill Bement1
1. Dept. of Zoology, University of Wisconsin-Madison, Madison, USA.
2. Dept. of Biochemistry, University of Wisconsin-Madison, Madison, USA.
#. Current address: Faculty of Life Sciences, University of Manchester, Manchester, UK.

The role of actomyosin in the mitotic spindle has long been a subject of controversy. Here we
present evidence that Myosin-10 (Myo10), an actin-based motor that binds microtubules,
participates with F-actin to regulate mitotic spindle function. Using Xenopus laevis early
embryos as an in vivo system to study mitosis, we show that Myo10 localises to the mitotic
spindle where it concentrates at the spindle poles from prometaphase through to the end of
telophase. Knockdown of Myo10 levels using an anti-sense morpholino approach reveals
that Myo10 is essential for 4 aspects of mitotic spindle function: proper spindle anchoring,
spindle length maintenance, spindle pole integrity, and progression through metaphase.
Furthermore, by using live imaging we show for the first time that highly dynamic F-actin
cables are present in the mitotic spindle and that these cables both surround the spindle and
extend between the spindle and the cortex. Remarkably, we find that although proper spindle
anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole
integrity is F-actin independent, while F-actin and Myo10 actually play antagonistic roles in
maintenance of spindle length. Our results show that Myo10 and F-actin play critical roles in
mitotic spindles and that Myo10 and F-actin have unexpected interdependencies.


The role of Xenopus Rx-L in photoreceptor cell determination
Hui-Yuan Wu1, Muriel Perron2 & Thomas Hollemann1
1
  Institut für Physiologische Chemie, Universität Halle-Wittenberg, D-06114
Halle/Saale, Germany
2
  Laboratoire «Gènes, Développement et Neurogenèse,» Université Paris XI, UMR
Centre national de la recherchescientifique (CNRS) 8080, 91405 Orsay, France.

Members of the Rx (retinal homeobox) gene family play vital roles during eye development in
vertebrates. Here, the second Rx-type gene, XRx-L, was identified from Xenopus. According
to a phylogenic analysis, all-known Rx-type genes could be grouped into four categories,
including the “invertebrate Rx” group, which contains all Rx genes from invertebrates, the
“classical vertebrate Rx” group, the “vertebrate Rx-Q50” group, and the “vertebrate Rx-like”
group to which XRx-L belongs.
The earliest expression of XRx-L can be detected in the presumptive eye area at late neurula
stage by WMISH. Suppression of XRx-L function in vivo by microinjection of Rx-L specific
antisense morpholino oligonucleotides impaired the formation of the photoreceptor layer and
reduced the expression of photoreceptor specific genes. Overexpression of XRx-L induced
ectopic expression of photoreceptor specific genes, but did not promote the proliferation of
retinal progenitor cells significantly. Targeted overexpression of XRx-L in developing
retinoblasts in vivo led to the increased fraction of photoreceptor cells at the expense of
amacrine and bipolar cells. Moreover, XRx-L was found to promote both rod and cone
photoreceptors, with a preference for rods. Our in vivo experiments also revealed that XRx-L
acts as a transcription activator.
Taken together, XRx-L, unlike XRx1, is required for the determination of retinal cell types,
especially photoreceptor cells, rather than to promote the proliferation of retinal progenitor
cells.

Vertebrate CASTOR is Required for Differentiation of Cardiac Precursor Cells at the Ventral
Midline

Kathleen S. Christine 1,2 and Frank L. Conlon 1,2,3
1
 Carolina Cardiovascular Biology Center, 2 Department of Biology, and 3 Department of Genetics,
University of North Carolina at Chapel Hill, Chapel Hill, NC
CASTOR (CST) is a zinc finger transcription factor initially identified for its role in maintaining stem cell
competence in the Drosophila dorsal midline. We have shown that Xenopus CST is expressed in the
developing heart and is required for the vertebrate cardiogenesis. In CST-depleted embryos,
cardiomyocytes at the ventral midline fail to differentiate and are maintained as cardiac progenitors.
Our fate mapping studies reveal that this ventral midline population of cardiomyocytes has a unique
fate ultimately giving rise to cardiomyocytes ib outer curvature of the heart; however, CST-depleted
midline cells over-proliferate and remain a coherent population of nonintegrated cells positioned on the
outer wall of the ventricle. These midline-specific requirements for CST suggest the regulation of
cardiomyocyte differentiation is regionalized along a dorsal-ventral axis and that this patterning occurs
prior to heart tube formation. To further characterize the molecular pathways by which CST acts, we
are taking a proteomics based approach to identify CST post translational modification and purify CST
protein complexes from Xenopus tropicalis.



Fus, a multifunctional RNA-binding protein, is necessary for FGF dependent gene
expression, gastrulation, and pre-mRNA splicing.
Darwin S. Dichmann and Richard M. Harland.

University of California, Berkeley; Department of Molecular and Cell Biology, 571 Life
Sciences Addition, Berkeley, CA 94720-3200



We isolated the multifunctional RNA-binding protein Fus in an expression cloning screen for
novel gene activities. Fus belongs to the FET-family (Fus, EWS, TAF15) of atypical RNA-
binding proteins containing transcriptional activation domains that are frequently translocated
in liposarcomas. Fus knockdown results in a complete absence of blastopore formation and
gastrulation, followed by cell dissociation and embryo death at stage 12.5. Gene expression
analysis shows that all germ layers form, show dorsal-ventral polarity, but fail to express
FGF-dependent genes such as brachyury and endodermin. In addition, eomesodermin, but
not brachyury, can be induced by Activin mRNA injection in embryos where Fus has been
knocked down, suggesting a specific defect in FGF-dependent gene induction. RT-PCR
analysis show inclusion of specific introns in key transcripts in the FGF signaling pathway
including fgf8 and fgfr2, suggesting a possible mechanism for the observed inhibition of FGF
target genes. To determine the mechanism resulting in the later cell detachment we analyzed
transcript integrity in members of the cadherin family and find widespread intron inclusion in
e-cadherin whereas c- and xb-cadherin transcripts are normal. These results demonstrate a
novel function of Fus as a regulator of specific pre-mRNA splicing evens in the context of
specific signaling pathways. Future global strategies will be discussed.
SAGE analysis of dorsal and ventral transcriptome of Xenopus tropicalis gastrula
Faunes, F.1,3, Sanchez, N.1,3, Melo, F.2,3, Larrain, J.1,3.
1. Dep. of Cell and Molecular Biology. 2. Dep. of Molecular Genetics and Microbiology.
3. Catholic University of Chile.

        Xenopus has been a favourable model for studying gene function during early
embryonic development. Substantial progress has been made in the identification of genes
and molecular mechanisms involved in dorsoventral patterning of Xenopus embryos.
However, all these pre-genomic approaches were generally biased to the detection of a
limited group of transcripts. In addition, global approaches in several species have
demonstrated that transcriptomes are more complex than expected. At present, genomic
approaches can be performed in Xenopus tropicalis because its diploid genome sequence is
available.
        In this work, we used the global approach SAGE to identify novel genes involved in
dorsoventral patterning of Xenopus embryos. SAGE permits a qualitative and quantitative
analysis of the transcriptomes and the detection of novel transcripts. SAGE libraries were
prepared from dorsal and ventral explants of Xenopus tropicalis gastrula and 30,000
tags/library have been obtained.
        Here, we present the comparative analysis of these libraries. We performed tag-
mapping by using bioinformatics (with both genome sequence and known transcripts) and an
experimental approach (reverse SAGE). It’s important to note that all tags from known genes
involved in dorsoventral patterning present the correct distribution and we have 125 tags with
significant difference between both libraries. We have verified differential expression by in
situ hybridization for transcripts with the highest SAGE difference between dorsal and ventral
libraries. These novel transcripts identified will be used for functional studies. This is the first
SAGE experiment in Xenopus tropicalis and we expect to find novel genes involved in
dorsoventral patterning.
Acknowledgements: CONICYT (Chilean Government) and Faculty of Biological Sciences, P
Catholic University of Chile.
Inductive signals patterning the pronephric mesoderm. Caroline V. White and Peter D. Vize.
Department of Biological Sciences, University of Calgary, Alberta T2N 1N4, Canada

Inductive signaling is a crucial first step in the process of organogenesis. Molecular signals
secreted from a source cell population affect the differentiation and specialization of target cell
populations, initiating organization into functioning organs. The process of organogenesis is
probably best understood in the eye, heart and kidney, where the genetic and molecular
interactions required for proper organ formation are well understood. Despite a thorough
understanding of the target genes and factors regulated in response to inductive signaling in
these organs, little is known about the identities or sources of inductive signals. The Xenopus
laevis kidney provides an excellent model for studying inductive signaling during organogenesis
as its embryonic form, the pronephros, is comprised of a single functional unit or nephron, and
the molecular pathways involved in pronephros formation are well conserved in the development
of all kidney forms in higher vertebrates. We are interested in determining the tissues
responsible for producing inductive signals responsible for initiating kidney development.
Previously, most studies have focused on the relationship between potential inducing tissues
and formation of the pronephric tubules, one of the three functional components of the nephron,
and have largely overlooked the other two components, the glomus and duct. The tubules,
glomus and duct are capable of forming independently of one another and are patterned in
response to different signals, making it likely that these components respond to signals from
different source tissues. We will bridge this gap by investigating the formation of all three
components of the pronephric nephron in the presence or absence of potential inducing tissues.
Determining the source tissues patterning the pronephric kidney is an important step toward
identifying the inductive signals they secrete, and thus elucidating their mechanisms of
downstream signaling.

				
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