RETINAL STEM CELL PROLIFERATION

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					                 CELLULES SOUCHES ET NEUROGENESE DE LA RETINE
                     RETINAL STEM CELLS AND RETINOGENESIS
                           Research projects 2010-2013

FIRST AXIS OF RESEARCH: RETINAL STEM CELL PROLIFERATION
SCIENTIFIC BACKGROUND, OBJECTIVES AND METHODS
         The molecular characterization of retinal stem cell identity and properties remains
limited, partly due to the relatively recent discovery of these cells in mammals (reviewed in
Perron et al., 2000). The central goal of our project is to gain insight into the molecular
regulatory network controlling retinal stem cell behaviour, i.e. self-renewal, proliferative
capacity and multipotency. Because retinal stem cell niche is sharply defined in Xenopus
tadpoles within the ciliary marginal zone (CMZ), this model system offers an exceptional tool
to perform in vivo investigations in this field.
       Hedgehog (Hh), Wnt and Notch signallings emerged as critical regulators of neural
stem cell maintenance in the adult brain (reviewed in Shi et al., 2008). However, their
respective contribution as well as potential cross-talks are far from being understood as both
synergistic or antagonist effets have been reported so far (Day et Yang, 2008; van den Brink
et al., 2004; Ulloa et al., 2007; Alvarez-Medina et al., 2008). In the continuity of our previous
studies, the ambition of our project is now to fill a gap in the field of retinal stem/precursor cell
biology, by investigating functional interactions established by Hedgehog, Wnt and Notch
signalling pathways. This will be explored in a physiological context at embryonic and post-
embryonic stages, and in regenerative conditions following retinal damage. Besides, in order
to identify new retinal stem cell markers, we recently initiated and will pursue a large scale in
situ hybridization screen. The expected outcome is to provide novel insight into the retinal
stem cell molecular signature and to reveal key intrinsic factors that will be subjected to
functional characterization. Our experimental strategy relies heavily on gain and loss of
function experiments through the use of pharmacological and/or genetic tools. Of note, we
have extensive experience with the main technical aspects of this project (microinjection, in
vivo lipofection, electroporation, transgenesis, …). Surgical retinal ablation studies will benefit
from the expertise of our collaborator, T. Hollemann (Germany). In addition, a conditional
targeted cell ablation method based on the NTR/Mtz system (Curado et al., 2008) is currently
under development in our lab.
PROPOSED WORK PLAN
 Task1. Functional interactions between Hedgehog, Wnt and Notch signalling in the
control of retinal stem cell behaviour
      To unravel the potential
cross talks between Hh, Wnt
and Notch signalling pathways
in the retina, we will first
evaluate whether perturbing
one pathway has an impact on
the activity of the two other one
in the CMZ. This will be tested
by monitoring transcriptional
target expression or using GFP
reporter constructs. Second,
cell   proliferation    will    be
analyzed                 following
simultaneous inhibition or activation of two pathways or when one pathway is activated while
another one is blocked. These experiments should reveal epistatic relationships as well as
synergistic or antagonistic effects of these signaling pathways on retinal cell proliferation. Our
preliminary data suggest that Hh and Wnt pathways establish opposite gradients within the
CMZ and might antagonize each other (Fig1). These promising results are under further
investigations to support our present working model.
      Finally, Hh, Wnt and Notch involvement an interactions in retinal stem cell biology will
be investigated during the time course of neural tissue regeneration following ablation of the
retina.


 Task2. Characterization of retinal stem cell
specific factors: a candidate gene approach
Functional characterization of the bHLH-O repressors
XHairy1 and XHairy2
        We recently identified XHairy1 and XHairy2
(orthologs of human Hes1 and Hes4, respectively) as
being specifically expressed in the stem cell-
containing region of Xenopus retina. Preliminary
experiments revealed that XHairy1/2 gain of function
inhibits differentiation of retinal precursor cells by
slowing down cell cycle speed and preventing cell
cycle exit (Fig2). Our current model proposes that
these factors contribute to the maintenance of retinal
stem cells by keeping them in a undifferentiated and
slowly proliferative state. Whether these factors might
constitute targets of Notch, Hh or Wnt signalling is
under investigations.

Functional characterization of the RNA binding
proteins Msi1 and Msi2
        Two other genes recently identified in the lab, Musachi1 (Msi1) and Musachi2 (Msi2),
encode RNA binding proteins and were found to be expressed in retinal stem cells. Msi
genes are well-known brain stem cell markers (reviewed in Okano et al., 2005 ; MacNicol et
al., 2008). Our promising preliminary experiments indicate that Msi1/2 are involved in the
control of retinal cell proliferation. Importantly, we also found that Msi misexpression induces
                                                          tumour formation in the epidermis
                                                          (Fig3). Preliminary data indicate that
                                                          these skin tumours express stem cell
                                                          markers (Fig3), and may form as a
                                                          consequence of Hh signalling activation.
                                                          These results constitute a fertile ground
                                                          for further characterization of the
                                                          mechanisms sustaining Msi effects on
                                                          retinal    cell    proliferation      and
                                                         oncogenesis.

Task 3. Identification of novel genes expressed in retinal stem cells
       Since very few genes have been discovered so far in retinal stem cells, we undertook
a transcriptomic approach to identify novel specific markers. We took advantage of the
Xenopus retina that offers the possibility to visualize the stem cell niche, to set up the basis
of a large-scale in situ hybridization screen. Beside the identification of retinal stem marker,
this project should generate a large collection that should be of extraordinarily unique value
not only for our study but also for other projects. Therefore, our results will be made public to
help the whole research community. The second phase of this screen project will consist in
further analysis of chosen candidates. In particular, they will be systematically tested as
potential target of the Notch, Wnt or Hh signalling pathways.

SECOND AXIS OF RESEARCH: RETINAL PROGENITOR DETERMINATION
        A great challenge in neurobiology is to identify intrinsic factors that control neural
progenitor cell destiny. Behind the simplicity of the retina, encompassing only six major cell
types, lies a huge diversity of retinal subtypes forming a complex and subtle structure.
Although much progress has been done during the last decade in understanding intrinsic
cues governing the specification of the major neural cell types in the retina, the molecular
mechanisms that generate the phenotypic diversity of retinal subtypes in term of
neurotransmitter content remains largely unknown.
        In the continuity of our work on Ptf1a, we now aim at dissecting the molecular network
controlling GABAergic and glutamatergic neuron production. A few candidate genes have
attracted our interest such as Xash1 (ortholog of Mash1), Gsh1/2, Lbx1, Tlx1 and Tlx3. We
already have preliminary data strongly supporting a role for Xash1 in the specification of
GABAergic neurons in the retina within the Ptf1a pathway. In addition, we are planning to
carry out a large scale functional screen in collaboration with K. Henningfeld and T. Pieler
(Germany) to isolate novel candidates. This project should significantly contribute to uncover
the genetic network governing GABAergic/glutamatergic determination in the retina.

MILESTONES
      Together, our project should provide new insights into the molecular mechanisms
sustaining retinal stem cell proliferation and maintenance and controlling the specification of
retinal subtype identity. All of our promising preliminary results, our expertise in the field, and
the novel approaches we have recently set up, render this project innovative and feasible. In
addition, the expected outcome should have relevance for cancerology and for the rational
design of therapeutical strategies involving retinal stem cells.

REFERENCES
Alvarez-Medina, R., Cayuso, J., Okubo, T., Takada, S., and Marti, E. (2008). Wnt canonical pathway restricts
      graded Shh/Gli patterning activity through the regulation of Gli3 expression. Development 135, 237-247.
Curado, S., Stainier, D.Y., and Anderson, R.M. (2008). Nitroreductase-mediated cell/tissue ablation in zebrafish: a
      spatially and temporally controlled ablation method with applications in developmental and regeneration
      studies. Nat Protoc 3, 948-954.
Day, T.F., and Yang, Y. (2008). Wnt and hedgehog signaling pathways in bone development. J Bone Joint Surg
      Am 90 Suppl 1, 19-24.
MacNicol, A.M., Wilczynska, A., and MacNicol, M.C. (2008). Function and regulation of the mammalian Musashi
      mRNA translational regulator. Biochem Soc Trans 36, 528-530.
Okano, H., Kawahara, H., Toriya, M., Nakao, K., Shibata, S., and Imai, T. (2005). Function of RNA-binding protein
      Musashi-1 in stem cells. Exp Cell Res 306, 349-356.
Perron, M., and Harris, W.A. (2000). Retinal stem cells in vertebrates. Bioessays 22, 685-688.
Shi, Y., Sun, G., Zhao, C., and Stewart, R. (2008). Neural stem cell self-renewal. Crit Rev Oncol Hematol 65, 43-
      53.
Ulloa, F., Itasaki, N., and Briscoe, J. (2007). Inhibitory Gli3 activity negatively regulates Wnt/beta-catenin
      signaling. Curr Biol 17, 545-550.
van den Brink, G.R., Bleuming, S.A., Hardwick, J.C., Schepman, B.L., Offerhaus, G.J., Keller, J.J., Nielsen, C.,
      Gaffield, W., van Deventer, S.J., Roberts, D.J., et al. (2004). Indian Hedgehog is an antagonist of Wnt
      signaling in colonic epithelial cell differentiation. Nat Genet 36, 277-282.

				
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posted:10/18/2011
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