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									                                         Welcome

Welcome to Developmental & Perinatal Biology 2006. This is the 10th Annual Exchange in
developmental and perinatal biology between The University of Toronto and The Karolinska
Institute. The research course has been developed to provide a broad based training for graduate
students, research fellows and clinical fellows in the area of developmental biology from both basic
science and clinical perspectives. The workshop combines a lecture/seminar program with an active
research component. The course is also offered as a component of a Graduate course (PSL1480H)
at the University of Toronto and as a Graduate course at the Karolinska Institute.

The 2005 course was held in August in Stockholm and was hosted by the Karolinska Institute;
organized by Professors Hugo Lagercrantz, Ola Hermanson and Thomas Ringstedt. It was attended
by 4 faculty and 15 trainees from The University of Toronto. From the attendance and success in
previous courses, it has been clear that there is great interest in this type of summer course. This
year we have experienced similar enthusiasm, with 4 faculty and 14 trainees attending from
Sweden, 2 faculty and 5 trainees from the U.K., along with trainees from The University of
Toronto. In addition, 16 faculty from across The University of Toronto, as well as one guest
lecturer from McGill University, will be involved in lecture and practical tuition.

The organization of this type of course requires a considerable input of energy. Therefore, we
would like to take this opportunity to thank those on the organizing committee for helping to put the
exciting course program together, and to Professor Thomas Ringstedt for co-ordinating the Swedish
side of the exchange. We would also like to thank Jenny Katsoulakos, Bev Bessey and Sophie
Petropoulos who have provided invaluable organizational support. Finally, we would like to
express our gratitude to our sponsors, many of whom have provided continuous support over the
last 10 years, and who have made Developmental & Perinatal Biology 2006 possible.

Please accept our warmest welcome to what we hope will be an exciting academic and social
experience.




       Dr. S. G. Matthews                                   Dr. S. J. Lye
       Professor                                            Professor
       Physiology, Ob-Gyn & Medicine                        Ob-Gyn, Physiology & Medicine




                                                    1            Developmental & Perinatal Biology 2006
Local Organizing Committee:
Steve Matthews (Chair)
Lee Adamson
Bev Bessey
John Challis
Jenny Katsoulakos
Steve Lye
Sophie Petropoulos
Sue Quaggin
Janet Rossant
Neil Sweezey


Co-Sponsors:
Hospital for Sick Children, Research Training Centre
Samuel Lunenfeld Research Institute, Mount Sinai Hospital
Department of Obstetrics & Gynaecology, University of Toronto
Department of Paediatrics, University of Toronto
Department of Physiology, University of Toronto
Faculty of Medicine, University of Toronto


Location:
Registration:    16th August – Outside Room 3163, Medical Sciences Building,
                 University of Toronto


Lectures:        16th-23rd August, Room 3163, Medical Sciences Building


Practical Workshops:         Laboratories located in the Medical Sciences Building, Samuel
                             Lunenfeld Research Institute, Mount Sinai Hospital, Ontario Cancer
                             Institute and Hospital for Sick Children Research Institute


Social Activities:    16th August   -      Welcome Reception, Music Room, Hart House ~7pm
                      18th August   -      Pub Night
                      19th August   -      Day Trip to Niagara Falls & Niagara-on-the-Lake
                      22nd August   -      BBQ on Toronto Island




                                                  2             Developmental & Perinatal Biology 2006
FACULTY:

University of Toronto:

                         Department                                   Location

Dr. Lee Adamson          Ob/Gyn                        Samuel Lunenfeld Research Institute
Dr. John Challis         Physiology / Ob/Gyn           University of Toronto
Dr. Leigh Coultas        PDF – Dr. Rossant‟s Lab       Hospital for Sick Children
Dr. Carolyn Dunk         Res. Assoc. – Dr. Lye‟s Lab   Samuel Lunenfeld Research Institute
Dr. Robert Jankov        Paediatrics                   Sunnybrook/Women‟s College HSC
Dr. John Kingdom         Ob/Gyn                        Mount Sinai Hospital
Dr. Stephen Lye          Ob/Gyn / Physiology           Samuel Lunenfeld Research Institute
Dr. Stephen Matthews     Physiology                    Faculty of Medicine, MSB
Dr. Michael Meaney*      Psych./Neurology              Douglas Hosp. Res. Centre, Montreal
Dr. Mira Puri            Medical Biophysics            Sunnybrook/Women‟s College HSC
Dr. Sue Quaggin          Medicine                      Samuel Lunenfeld Research Institute
Dr. Janet Rossant        Med. Gen. & Micro.            Hospital for Sick Children
Dr. Ian Scott            Develop. & Stem Cell Biol.    Hospital for Sick Children
Dr. Cheryle Seguin       PDF – Dr. Rossant‟s Lab       Hospital for Sick Children
Dr. Marla Sokolowski     Zoology                       University of Toronto, Mississauga
Dr. Neil Sweezey         Paediatrics                   Hospital for Sick Children
Dr. Keith Tanswell       Paediatrics                   Hospital for Sick Children


* Guest Lecturer


Karolinska Hospital/Institute:
Dr. Ola Hermanson
Dr. Hugo Lagercrantz
Dr. Thomas Ringstedt
Dr. Karl Tryggvason


University of Manchester:
Dr. Ian Crocker
Dr. Rebecca Lee Jones




                                                3           Developmental & Perinatal Biology 2006
Wednesday, 16th August

8:30    Registration                Medical Sciences Building, Outside Room 3163
8:50    Welcome/Introduction        Dr. Stephen G. Matthews
                                    Chair, International Exchange Program for Developmental and
                                    Perinatal Biology, University of Toronto
                                    Dr. John Challis
                                    Vice-President, Research and Associate Provost,
                                    University of Toronto

                               Pregnancy and Birth
                             (Medical Sciences Building, Room 3163)
                                 Co-ordinator: Dr. Steve Lye


9:00    Embryo implantation and the establishment of pregnancy
                                                                                  Dr. R. Lee Jones

9:40    Extravillous trophoblast and the development of the uteroplacental circulation
                                                                                      Dr. C. Dunk

10:20 Coffee


10:40 The villous placenta – development and dysfunction
                                                                                     Dr. I. Crocker

11:20 Molecular pathology of IUGR and pre-eclampsia
                                                                                    Dr. J. Kingdom

Trainee Presentations (Orals 1-3)

12:00 Placental multidrug resistance phosphoglycoprotein in mouse pregnancy:      Fetal
      protection.                                              Sophia Petropoulos1

12:15 Placental expression of the system A isoforms SNAT1, 2 and 4 and their functional
      importance for amino acid transport and fetal growth.     Michelle Desforges2

12:30 Reactive oxygen species alter vascular responses of chorionic plate small arteries.
                                                                        Tracey Mills3
13:00 Lunch

14:00-17:00    Research Workshop

19:00          WELCOME RECEPTION: Music Room, Hart House, 7 Hart House Circle, UofT



                                                   4           Developmental & Perinatal Biology 2006
Thursday, 17th August


                    Pulmonary & Renal Development
                            (Medical Sciences Building, Room 3163)
                               Co-ordinator: Dr. Neil Sweezey


9:00   Renal development
                                                                                   Dr. K. Tryggvason

10:00 Coffee


10:20 Chronic lung disease of prematurity
                                                                                      Dr. K. Tanswell

11:10 Pulmonary blood pressure
                                                                                        Dr. R. Jankov


Trainee Presentations (Orals 4-6)


12:00 Role of IL-1 in the rat neonatal model of acute lung injury mediated by oxygen toxicity.
                                                                       Ben-Hur Johnson4

12:15 Cross talk among prolactin and dopamine in renal tubular cells.
                                                                            Susanne Crambert5

12:30 Localization of the dopamine transporter in renal tissue.
                                                                            Agneta Sjöberg6

12:45 Respiratory and cardiovascular dysfunction in Rett syndrome - autonomic characteristics
      and treatment based on long term monitoring.                    Malin Rohdin7


13:00 Lunch


14:00-17:00    Research Workshop




                                                  5               Developmental & Perinatal Biology 2006
Friday, 18th August


              Cardiovascular Development and Function
                             (Medical Sciences Building, Room 3163)
                               Co-ordinator: Dr. Lee Adamson


9:00   Specification and differentiation of endothelial and hematopoietic cell lineages in the
       early vertebrate embryo
                                                                                       Dr. M. Puri

9:40   The developing vasculature and its role in organogenesis
                                                                                     Dr. L. Coultas

10:20 Coffee


10:40 Fishing out new modifiers of cardiac development
                                                                                        Dr. I. Scott

11:20 Functional development of the cardiovascular system
                                                                                   Dr. L. Adamson

Trainee Presentations (Orals 7-10)

12:00 Doppler echocardiographic and electrocardiographic atrioventricular time intervals in
      newborn infants: Evaluation of techniques for surveillance of fetuses at risk for
      congenital heart block.                                      Gunnar Bergman8

12:15 Characterization of the uPA-mediated cleavage of PDGF-DD.
                                                                         Monika Ehnman9

12:30 Identification and functional validation of genes selectively expressed in vascular
      endothelial cells.                                           Elisabet Wallgaard10

12:45 Studying phase singularities, activation rates (dominant frequencies), and conduction
      block during human ventricular fibrillation                      Nimalan Thavandiran11

13:00 Lunch (Lobby of the J.J.R. MacLeod Auditorium – MSB 2158)

13:45 GROUP PHOTO (please assemble outside on steps of MSB facing King‟s College Circle)

14:00-17:00    Poster Session (Lobby of the J.J.R. MacLeod Auditorium – MSB 2158)

19:00-Late     “Pub Night”


                                                   6           Developmental & Perinatal Biology 2006
Monday, 21st August


                                Neurodevelopment
                           (Medical Sciences Building, Room 3163)
                             Co-ordinator: Dr. Steve Matthews


9:00   CNS development: The developing mind
                                                                              Dr. H. Lagercrantz

9:40   Leave no histone unturned: Transcriptional and epigenetic regulation of neural
       development
                                                                             Dr. O. Hermanson

10:20 Coffee


10:40 Axonal guidance and nerve growth factors
                                                                                 Dr. T. Ringstedt

11:20 Fetal environment and the developing brain
                                                                                 Dr. S. Matthews


Trainee Presentations (Orals 11-13)


12:00 Protein nitration causes neuronal death following traumatic brain injury.
                                                                      Michael Jones12

12:15 Expressional differences in endogenous control genes and in genes controlling
      myelination, cell death/survival and signaling in motor and frontal cortices of human
      chronic alcoholics.                                           Sofia Johansson13

12:30 SIRT1 deacetylase-dependent and –independent effects of resveratrol and red wine on
      neural stem and neuroblastoma cells.                        Karolina Wallenborg14


13:00 Lunch


14:00-17:00    Research Workshop




                                                 7           Developmental & Perinatal Biology 2006
Tuesday, 22nd August


                Embryo Patterning and Organogenesis
                           (Medical Sciences Building, Room 3163)
                     Co-ordinators: Drs. Janet Rossant and Sue Quaggin


9:00    Molecular genetics of extraembryonic development
                                                                                 Dr. J. Rossant

9:40    Embryonic stem cells – derivation and uses
                                                                                  Dr. C. Seguin

10:20 Coffee


10:40 Kidney development
                                                                                Dr. S. Quaggin



Trainee Presentations (Orals 14-16)


12:00 Maternal endowment of Bcl-2 family members in human and murine oocytes.
                                                                Ingrid Lai15

12:15 Podocyte-specific knock-out of mammalian Target of Rapamycin (mTOR): Its role in
      glomerular development, function and pathology.              Aarti Paltoo16

12:30 A genetic approach for establishing novel stem-cell lines from lineages of
      postimplantation stage mouse embryos.                 Ridham Desai17


13:00 Lunch


14:00-17:00    Research Workshop


18:00          BBQ on Toronto Island




                                                     8     Developmental & Perinatal Biology 2006
Wednesday, 23rd August


          Developmental Origins of Health and Disease
                           (Medical Sciences Building, Room 3163)
                              Co-ordinator: Dr. John Challis


9:00   Gene by environment interactions: Coupling foraging behaviours to the environment via
       cGMP dependent protein kinases (PKG)
                                                                           Dr. M. Sokolowski

9:40   Programming and preterm birth
                                                                                   Dr. J. Challis

10:20 Coffee


10:40 Maternal care, gene expression and epigenetic programming of the HPA stress response
                                                                             Dr. M. Meaney



Trainee Presentations (Orals 17-19)


12:00 PGE2 concentration and COX-2 protein expression in human amnion epithelial and
      mesenchymal cells treated with meloxicam, arachidonic acid and prostaglandin E2.
                                                                     Valerija Rac18

12:15 Hypothalamic adiponectin receptor gene expression is modulated in adult mouse
      offspring exposed to a maternal diet high in fat and protein during pregnancy and
      lactation.                                                  Dyan Sellayah19

12:30 Chronic maternal adversity and offspring growth, behaviour and hypothalamo-pituitary-
      adrenal (HPA) function: male vulnerability.                  Jeff Emack20


       Close of Course

13:00 Lunch




                                                9           Developmental & Perinatal Biology 2006
Practical Workshops:
1.       Endocrine Gene Expression: High-Throughout Technologies for Translational
         Research
                                                                 Dr. Stephen Lye

Location:      Mount Sinai Hospital

Leaders:       Dr. Jim Woodgett, Dr. Lorne Taylor, Dr. Ian Crocker, Dr. Rebecca Lee Jones,
               Dr. Jonathan Perkins, Dr. Alessandro Datti

In these sessions we will introduce the different approaches and techniques used in today‟s high-
throughput technologies considering:

Time:

Day 1 (August 16 & 21 from 2:00-5:00):

        Transcriptomics – Gene Arrays
        SNP Analysis
        ChIP on chip

Day 2 (August 17 & 22 from 2:00-5:00):

        Metabonomics
        Proteomics
        Automated technologies

Site Visits

We will be visiting the Ontario Cancer Institute microarray facility (www.microarrays.ca) and
Robotics laboratories of the SLRI (www.mshri.on.ca/robotics).




                                                 10            Developmental & Perinatal Biology 2006
2.       Genetic Manipulation of the Embryo                                   Dr. Janet Rossant

This workshop has some hands-on experience as well as demonstrations of the techniques involved
in generating and analysis of genetically altered mice

Leaders:
Sophia George (Sophia@mshri.on.ca)
Vera Eremina (eremina@mshri.on.ca)

Location:
Research Annex, Mount Sinai Hospital Transgenic Room
(meet at The Second Cup in the hospital lobby at 1:45 pm)

Time:
Group 1: Day 1 - August 16 (2 pm – 5 pm); Day 2 - August 17 (2 pm – 5 pm)
Group 2: Day 1 - August 21 (2 pm – 5 pm); Day 2 - August 22 (2 pm – 5 pm)

Program:

Day 1:

Introduction: Current technologies in mouse genome manipulation

Demonstrations
Chimeric animals, GFP, RFP animals
Xgal stained embryos
Whole mount in situ hybridization
Skeletal preps

Hands-on experiments
Dissection of postimplantation stage embryos (E9.5-10.5)
GFP examination
Dissection of organs from E15.5 embryos

Day 2:

Demonstrations
Glomeruli-specific LacZ expression and in situ

Hands-on experiments
Glomeruli isolation from Podocin-CFP transgenic mice

Discussion




                                                 11           Developmental & Perinatal Biology 2006
3.       New Techniques in Mouse Physiology                                       Dr. Lee Adamson

Location:       Mount Sinai Hospital

Mice are useful models for assessing the long-term pathophysiologic effects of developmental
defects whether caused by abnormal environmental influences or genes. In this lab, students will
see living mouse embryos in utero using a newly-developed, high-resolution, 30-MHz ultrasound
scanner. In adult mice, they will measure cardiac performance using Doppler ultrasound, measure
blood pressure and heart rate with a tail-cuff plethysmograph, and collect blood samples for
hematologic analysis. Results from mice deficient in nitric oxide synthase (NOS) will be compared
to controls to determine the pathophysiologic effects of reduced NOS activity in adult mice.


4.       Lung Physiology/Cell Biology                     Drs. Neil Sweezey and Keith Tanswell

Location:       McMaster Building, Hospital for Sick Children

The afternoon session for pulmonary development will include the following experiments:

        Fetal rat lung primary culture
        Adult rat lung primary culture
        Sectioning – paraffin and cryo-blocks
        Laser capture dissection
        Discussion period
        Ussing chamber
        Mouse mechanics and ventilation
        Stretch systems
        Smooth muscle cells
        Discussion Period



5.       Imaging Technologies                                               Dr. Steve Matthews

Location:       Various (please meet in MSB Stone Lobby at 2pm)

Students will gain experience in the use and theory of:

Microscopy:            Confocal and EM
                       Image analysis techniques

MRI:                   Fetal and developing brain

Ultrasound:            Doppler ultrasound
                       3-D Ultrasound
                       Fetal endoscopy and operative procedures


                                                    12            Developmental & Perinatal Biology 2006
Research Workshop Assignments:


                                 16/17th August     21/22nd August

Desai           Ridham                3                     4
DiGiovanni      Valeria               3                     2
Emack           Jeff                  1                     5
Fornaro         Enrica                4                     3
Galtseva        Alevtina              4                     5
Gill            Paul                  2                     3
Jain            Venu                  5                     1
Jayasuriya      Kusala                3                     1
Johnson         Ben-Hur               1                     2
Jones           Michael               3                     5
Juneja          Subhash               2                     1
Kapoor          Amita
Lai             Ingrid                3                     2
Paltoo          Aarti                 3                     5
Petropoulos     Sophie                5                     3
Rac             Valerija              1                     4
Thavandiran     Nimalan               1                     4


Bergman         Gunnar                5                     4
Carlsson        Jakob                 4                     1
Crambert        Susanne               3                     5
Desforges       Michelle              5                     2
Ehnman          Monika                2                     3
Hollis          Lisa                  1                     4
Johansson       Sofia                 4                     2
Mills           Tracey                4                     3
Nisancioglu     Maya                  2                     3
Olson           Linus                 1                     5
Rohdin          Malin                 2                     4
Sandberg        Magnus                3                     2
Sellayah        Dyan                  5                     3
Sjöberg         Agneta                5                     4
Södersten       Erik                  2                     1
Sundqvist       Johanna               1                     5
Tower           Clare                 5                     1
Wallenborg      Karolina              2                     1
Wallgard        Elisabet              4                     2




                                     13           Developmental & Perinatal Biology 2006
                                     ABSTRACTS

                                           - Orals -
O1
Sophie Petropoulos, G.M. Kalabis, W. Gibb and S.G. Matthews. Placental multidrug resistance
phosphoglycoprotein in mouse pregnancy: Fetal protection.

O2
Michelle Desforges, S.L. Greenwood, J.D. Glazier, M. Westwood, M. Constancia and C.P. Sibley.
Placental expression of the system A isoforms SNAT1, 2 and 4 and their functional importance for
amino acid transport and fetal growth.

O3
Tracey Mills, M. Wareing, S.L. Greenwood, C.P. Sibley and P.N. Baker. Reactive oxygen species
alter vascular responses of chorionic plate small arteries.

O4
Ben-Hur Johnson and A. Keith Tanswell. Role of IL-1 in the rat neonatal model of acute lung
injury mediated by oxygen toxicity.

O5
Susanne Crambert, Fernando Ibarra, Peter Hansell and Ulla Holtbäck. Cross talk among prolactin
and dopamine in renal tubular cells.

O6
Agneta Sjöberg, Tove Tingvall and Ulla Holtbäck. Localization of the dopamine transporter in
renal tissue.

O7
Malin Rohdin, Miriam Katz-Salamon and Hugo Lagercrantz. Respiratory and cardiovascular
dysfunction in Rett syndrome – autonomic characteristics and treatment based on long term
monitoring.

O8
Gunnar Bergman, L-A. Jacobsson, M. Wahren-Herlenius and S-E. Sonesson.                   Doppler
echocardiographic and electrocardiographic atrioventricular time intervals in newborn infants:
Evaluation of techniques for surveillance of fetuses at risk for congenital heart block.

O9
Monika Ehnman and Ulf Eriksson. Characterization of the uPA-mediated cleavage of PDGF-DD.

O10
Elisabet Wallgaard, Mats Hellström, Liqun He, Erik Larsson, Mattias Kalén, Minoru Takemoto,
Noomi Asker, Linda Karlsson, Perry Hackett, Per Lindahl and Christer Betsholtz. Identification
and functional validation of genes selectively expressed in vascular endothelial cells.

                                                14            Developmental & Perinatal Biology 2006
O11
Nimalan Thavandiran, K. Nanthakumar, Sephane Massé, Rajesh Dhopeshwarkar, Elias
Sevaptsidis and John Asta. Studying phase singularities, activation rates (dominant frequencies),
and conduction block during human ventricular fibrillation.

012
Michael Jones, Hong-Shuo Sun and Mike Tymianski. Protein nitration causes neuronal death
following traumatic brain injury.

O13
Sofia Johansson, Clive Harper, Yasmin L. Hurd, Georgy Bakalkin and Tomas Ekström.
Expressional differences in endogenous control genes and in genes controlling myelination, cell
death/survival and signaling in motor and frontal cortices of human chronic alcoholics.

O14
Karolina Wallenborg, P. Vlachos, L. Huijbregts, B. Joseph and O. Hermanson. SIRT1 deacetylase-
dependent and –independent effects of resveratrol and red wine on neural stem and neuroblastoma
cells.

O15
Ingrid Lai and Andrea Jurisicova. Maternal endowment of Bcl-2 family members in human and
murine oocytes.

O16
Aarti Paltoo and Sue Quaggin. Podocyte-specific knock-out of mammalian target of rapamycin
(mTOR): Its role in glomerular development, function and pathology.

017
Ridham Desai, Gusztav Belteki and Andras Nagy. A genetic approach for establishing novel stem-
cell lines from lineages of postimplantation stage mouse embryos.

O18
Valerija Rac, I. Kamikovski, J. Challis and S.J. Lye. PGE2 concentration and COX-2 protein
expression in human amnion epithelial and mesenchymal cells treated with meloxicam, arachidonic
acid and prostaglandin E2.

O19
Dyan Sellayah, Kimberley D. Bruce, Chunli Wang, Frederick W. Anthony, Mark A. Hanson,
Christopher D. Byrne and Felino R.A. Cagampang. Hypothalamic adiponectin receptor gene
expression is modulated in adult mouse offspring exposed to a mataernal diet high in fat and protein
during pregnancy and lactation.

O20
Jeff Emack, Alice Kostaki, Dominique Walker and Stephen Matthews. Chronic maternal adversity
and offspring growth, behaviour and hypothalamo-pituitary-adrenal (HPA) function: male
vulnerability.



                                                   15            Developmental & Perinatal Biology 2006
O1
MULTIDRUG RESISTANCE IN THE MOUSE PLACENTA: FETAL PROTECTION

S Petropoulos1, GM Kalabis1, W Gibb4, SG Matthews1-3; Depts. of Physiology1, Ob-Gyn2 and
Medicine3, University of Toronto Depts. of Ob-Gyn, Cellular and Molecular Medicine4, University
of Ottawa

Background: Multidrug resistance genes (mdr1a and mdr1b) and their associated proteins, P-
glycoproteins (P-gps), have been identified in rodent tissues. Early studies suggest that Mdr1/P-gp,
present on the apical surface of placental syncitiotrophoblast cells, functions to reduce fetal
exposure to drugs and toxins present in maternal circulation. We have recently shown the there is a
marked decrease in the expression of placental Mdr1/P-gp in the human and mouse placenta with
advancing gestation which coincides to the decreasing levels progesterone seen in the latter species,
suggesting a regulatory role of progesterone. Further, progesterone has been shown to inhibit
Mdr1/P-gp efflux. Despite recent advancements in literature on Mdr1/P-gp, information concerning
the physiological function and regulation of placental Mdr1/P-gp is limited. In the present study,
we hypothesize that 1) Mdr1/P-gp mediated transplacental transfer changes with gestational age and
2) progesterone up-regulates placental Mdr1/P-gp expression and inhibits function.
Methods: 1: Pregnant FVB mice (gestational days (gds) 12.5, 15.5 and 18.5; n=5/gp) were injected
(s.c.) with [3H]digoxin (50μg/kg). Dams were euthanized 1h post injection. Maternal plasma, and
„fetal-units‟ (fetal membranes intact containing the fetus and amniotic fluid) were collected and
radioactivity determined. 2: Pregnant FVB dams (n=5/gp) were injected (s.c.) daily from gds14.5-
18.5 inclusive with either progesterone (16μg/kg in 100μl corn oil) or vehicle (100μl corn oil). On
gd18.5, [3H]digoxin was injected 2h after progesterone or vehicle injection. Drug distribution was
determined as above. Placentas (n=2/litter/gp) were collected and mdr1a/1b mRNA expression was
determined using real-time RT PCR.
Results: Using in vivo drug transfer experiments, we have demonstrated a significant increase in
transplacental transfer to the „fetal-unit‟ in late gestation (gd18.5) when compared to gd12.5
(P<0.01) and gd15.5 (P<0.01). No significant difference was observed in placental mdr1a/1b
mRNA expression between progesterone and vehicle treated dams. Transplacental transfer of
[3H]digoxin did not differ between the two groups.
Conclusions: Placental Mdr1/P-gp plays a critical role in the protection of the developing fetus.
This is especially pronounced earlier in gestation when placental Mdr1/P-gp is highly expressed.
Progesterone treatment did not appear to elicit a regulatory role on either expression or function of
placental Mdr1/P-gp. New knowledge in this area will enable the development of improved
strategies for fetal protection against the entry of potentially harmful maternal therapeutic agents
and environmental toxins.

(Supported by The Canadian Institute of Health Research)




                                                   16            Developmental & Perinatal Biology 2006
                                                                                                   O2
PLACENTAL EXPRESSION OF THE SYSTEM A ISOFORMS SNAT1, 2, AND 4 AND
THEIR FUNCTIONAL IMPORTANCE FOR AMINO ACID TRANSPORT AND FETAL
GROWTH
Michelle Desforges1, S.L. Greenwood1, J.D. Glazier1, M. Westwood1, M. Constancia2 and C.P.
Sibley1
1
  Division of Human Development, St Mary‟s Hospital, The Medical School, University of
Manchester, Manchester, UK
2
  Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, UK


Fetal growth is largely determined by the ability to access key nutrients such as amino acids from
the maternal circulation. During the second half of normal pregnancy fetal growth increases at a
faster rate than placental growth suggesting placental transport capacity must increase to meet rising
fetal nutrient demands. Data from human studies and animal models suggests the placental system
A transporter is particularly important in the supply of amino acids necessary for normal fetal
growth. There is evidence that system A activity in human placenta increases between first trimester
(FT) and term but the mechanisms responsible are unknown. Molecular characterisation of system
A reveals there are three isoforms (SNAT1, 2 and 4) with varying substrate specificities. We have
shown that mRNA expression for SNAT4 is significantly higher in early FT placenta compared to
term. In contrast, protein expression for this isoform is significantly higher at term compared to FT
suggesting complex regulatory mechansims may be in operation. SNAT1 protein expression also
increases between FT and term. Therefore, increased transporter protein expression could be
responsible for increased placental system A activity over gestation. The activity of this transporter
is reduced by 40-60% in placentas from human intrauterine growth restriction (IUGR). We have
preliminary data to show mRNA for the three system A isoforms is altered in these placentas. In a
recently developed mouse model of IUGR, placental system A activity is altered and this is
specifically associated with changes in mRNA expression for SNAT4. These data therefore raise
questions concerning the relative functional importance of the different isoforms of system A, in
particular SNAT4, in placental transport of amino acids. The aim of our current project is to test the
hypothesis that placental SNAT4 is functionally important in transporting amino acids required for
normal fetal growth. We will use an integrated molecular, cellular and whole animal approach to
determine: i) SNAT4 activity in human placenta, alone and in relation to that of SNAT1 and 2. ii)
the functional activity of SNAT4 in placenta over gestation and its regulation; iii) the role of
SNAT4 in amino acid supply to the fetus, using a mouse model of intrauterine growth restriction.




                                                    17            Developmental & Perinatal Biology 2006
                                                                                                O3
REACTIVE OXYGEN SPECIES ALTER VASCULAR RESPONSES OF CHORIONIC
PLATE SMALL ARTERIES

Tracey A. Mills, M. Wareing, S.L. Greenwood, C.P. Sibley and P.N. Baker

Division of Human Development, University of Manchester, Manchester, UK


Objectives: Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with raised
fetoplacental vascular resistance, the cause of which is unknown. Reactive oxygen species (ROS) are
implicated in vascular disease and are raised in PE/IUGR. We hypothesise that ROS (superoxide and
hydrogen peroxide) alter the vascular responses of chorionic plate arteries.

Methods: Placentas (n=8) were collected following normal pregnancies. Chorionic plate arteries
were prepared for wire myography. Paired vessels were incubated with xanthine (XA: 10 -4M) plus
xanthine oxidase (XO: 10mU/ml), to generate superoxide and hydrogen peroxide, or with vehicle
diluent throughout the experiment. After 10 min, the contractile response of the arteries to U46619
(thromboxane A2 mimetic; 10-10–10-6M) was determined. Endothelium-independent relaxation was
assessed in pre-constricted vessels (U46619 EC80) using sodium nitroprusside (SNP, nitric oxide
donor, 10-9–10-4M).

Results: XA/XO raised passive tension (2.89± 0.74 vs controls 0.43± 0.23; kPa mean±SE, p<0.05 t-
test). There was a left shift of the U46619 dose response curve with XA/XO (p<0.01; two-way
ANOVA) indicative of increased constriction (max tension 9.99±1.24 XA/XO vs 8.44± 0.82 control).
SNP-induced vasodilatation was enhanced by XA/XO (p<0.05, two-way ANOVA vs time control).

Conclusion: ROS generated by XA/XO increased chorionic plate artery basal tone and altered the
responses to agonists. We propose that ROS contribute to abnormal fetoplacental vascular resistance
in PE/IUGR.




                                                 18            Developmental & Perinatal Biology 2006
                                                                                                   O4

ROLE OF IL-1 IN THE RAT NEONATAL MODEL OF ACUTE LUNG INJURY
MEDIATED BY OXYGEN TOXICITY

Ben-Hur Johnson1 and A. Keith Tanswell2
1,2
   Departments of Paediatrics and Physiology, University of Toronto, Toronto, Canada; Lung
Biology Program, The Hospital for Sick Children Research Institute, Toronto, Canada
1
  Department of Newborn & Developmental Paediatrics, Sunnybrook Health Sciences Centre,
Toronto, Canada
2
  Division of Neonatology, The Hospital for Sick Children, Toronto, Canada


Bronchopulmonary Dysplasia (BPD), a common long-term complication associated with preterm
birth, developing in the most immature survivors of neonatal intensive care. The major pathologic
feature of BPD is a global reduction in alveolar number and surface area, consistent with an
inhibition or arrest of normal lung maturation and vascular development. A significant
inflammatory response of the small airways and interstitium of the lungs is also present, reflected in
the presence of neutrophils and macrophages which are immunoreactive for numerous cytokines
and humoral mediators. These proinflammatory cytokines, together with reactive oxygen species
(ROS) and mechanical ventilation, may be responsible for the acute neonatal lung injury.

Neonatal rats exposed to 60% O2 for 14 days develop patchy areas of interstitial thickening and
arrest of septation, consistent with human newborns with BPD. Previous experiments in our
laboratory have shown that just a few cytokines are up-regulated in these animals after exposure to
hyperoxia, including IL-1 and IL-1 and IL-18.

Hypothesis: The inflammatory process evident in the neonatal rat model of BPD is due in part to an
increased expression of IL-1 and IL-1, which results in an arrest of alveologenesis through a
ROS-mediated dysregulation of the expression of critical growth factors, such as FGF-7.

Methodology: The action of IL-1 was inhibited by daily injections of an IL-1-receptor antagonist
(IL-1Ra) in pups exposed to air or 60% O2 for 14 days, during the induction of the acute lung
injury. Lung injury was quantified by morphologic/morphometric analyses and immunolocalization
of involved inflammatory cells.

Preliminary results: Treatment of 60% O2-exposed pups with IL-1Ra reduced neutrophil influx,
and attenuated the 60% O2-mediated thickening of alveolar septa and reduction in secondary crests
and alveolar density associated with acute lung injury, consistent with a restoration of normal lung
development.

Preliminary conclusion: Under conditions of hyperoxia, IL-1 contributes significantly to the
development of acute neonatal lung injury, making it a valid target for intervention in BPD.




                                                    19            Developmental & Perinatal Biology 2006
                                                                                                  O5

CROSS TALK AMONG PROLACTIN AND DOPAMINE IN RENAL
TUBULAR CELLS

Susanne Crambert1, Fernando Ibarra1, Peter Hansell2 and Ulla Holtbäck1
1
    Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
2
    Department of Physiology, Uppsala University, Uppsala, Sweden


Background
Prolactin is a polypeptide involved in various actions in the body, including lactation,
osmoregulation, reproductive and parental behaviour. Prolactin is synthesized and secreted
primarily from lactotrophic cells of the anterior pituitary gland, and inhibited by the dopamine type
2 receptor (D2R). Prolactin is known to regulate transport of fluids across the plasma membrane, in
1959 Pickford and Phillips showed that prolactin have a crucial role in regulating salt and water
balance in fish migrating from salt to fresh water. Although prolactin affects sodium and water
transport across the plasma membrane and interacts with dopamine in the brain, its role in the
kidney is unclear. Here we examined the effect of prolactin and its interaction with the intrarenal
natriuretic hormone dopamine on proximal tubular Na+,K+-ATPase.

Methods
Confirmation of prolactin receptors in renal proximal tubuli and studies of phosphorylation on
Na+,K+-ATPase by protein kinase C (PKC) were made with conventional western blot techniques.
Na+,K+-ATPase activity was measured as ouabain sensitive ATP hydrolysis in microdisected
proximal tubular segments.

Results
Prolactin caused a dose dependent decrease in Na+,K+-ATPase activity. A maximum inhibitory
effect of 48% of control was observed at 1g prolactin/ml. The effect of prolactin was completely
abolished by a D1R antagonist but not a D2R antagonist. The renal D1R couples to adenylate
cyclase and the PKC signaling pathways. An inhibitor of adenylate cyclase or PKC significantly
attenuated the effect of prolactin. Prolactin induces phosphorylation of Na+,K+-ATPase by PKC.

Conclusion
We present prolactin as a potent natriuretic polypeptide, which acts by inhibiting tubular Na+,K+-
ATPase activity. The renal effects of prolactin require an intact intrarenal dopamine system and
activation of D1R.




                                                   20            Developmental & Perinatal Biology 2006
                                                                                                  O6
LOCALIZATION OF THE DOPAMINE TRANSPORTER IN RENAL TISSUE

Agneta Sjöberg, Tove Tingvall and Ulla Holtbäck

Woman and Child Health, Karolinska Institutet, Stockholm, Sweden


Dopamine, produced in renal proximal tubule cells, is a natriuretic hormone and has a key role in
the interactive regulation of salt balance. Animal models with genetic hypertension and patients
with essential hypertension have defects in the renal dopamine system. In spite of normal dopamine
levels, receptors and signaling molecules, dopamine is not able to increase natriuresis or decrease
Na, K-ATPase activity in renal tubule cells. In CNS, the activity of dopamine is regulated by a
dopamine specific transporter, DAT. DAT is a membrane bound protein, located on presynaptic
neurons and terminates dopaminergic neurotransmission primarily by reuptake of released
dopamine. We have found that DAT knock out mice develop hypertension and have a blunted
response to dopamine on Na, K-ATPase activity. Up to this date, no study is published whether
DAT is expressed in renal tissue.

We have found that mRNA DAT is expressed in renal tissue and predominantly in the cortex. Both
PCR analysis and in situ hybridisation support these results. The distribution of DAT protein by
IHC techniques mimicked the result from in situ hybridization, with DAT mainly expressed in renal
cortex. Subcellular fractionation followed by Western blot resulted in an anti-body specific band at
80 kDA in the cytosolic fraction. Interestingly, DAT is mainly found in the plasma membrane in
CNS at least in the synaptic cleft. Dopamine release via DAT is here regulated in a PKC-dependent
fashion. Whether regulation of DAT translocation and dopamine release also occurs in renal tissue
requires further investigation. One hypothesis is that dopamine it self, probably via PKC, triggers
DAT to translocate to the plasma membrane and release dopamine from intracellular vesicles.




                                                   21            Developmental & Perinatal Biology 2006
                                                                                                    O7
RESPIRATORY AND CARDIOVASCULAR DYSFUNCTION IN RETT SYNDROME –
AUTONOMIC CHARACTERISTICS AND TREATMENT BASED ON LONG TERM
MONITORING

Malin Rohdin, Miriam Katz-Salamon and Hugo Lagercrantz

Department of Women and Child Health, Astrid Lindgren Children's hospital, Neonatal research
Unit, Karolinska Institutet, Stockholm, Sweden


Rett syndrome is a progressive neurodevelopmental disorder with an abnormal function of the
cardiorespiratory system. The regulation of respiratory and cardiovascular function is a dynamic
process that changes with time, activity and state (sleep, awake). Therefore, it is of great importance
to characterize cardiorespiratory function during long term. Rett patients do not cope well with
unfamiliar environments and therefore, it is crucial to monitor the cardiorespiratory function in their
normal environment. The unique feature of this study is the continuous cardiorespiratory monitoring
of the Rett children at home, day and night during one week. Complementary investigations to
consider are polysomnography and Holter ECG. We will also compare the genotype with the
clinical phenotype, in particular the cardiorespiratory behaviour. Another objective with our study is
to simultaneously analyze the long term EEG activity and the cardiorespiratory regulation to better
distinguish epileptic from non-epileptic seizures.

All Rett children monitored so far revealed respiratory dysfunction and heart rate abnormalities
were seen in more than 50% of the children. Preliminary data indicate that long term and thorough
monitoring is crucial in studies of autonomic function in Rett patients. Our future plan is to establish
objective methods for assessments of the clinical status of the Rett patient, before and during
clinical trials and be able to accurately monitor changes over time.




                                                     22            Developmental & Perinatal Biology 2006
                                                                                                    O8
DOPPLER ECHOCARDIOGRAPHIC AND ELECTROCARDIOGRAPHIC ATRIO-
VENTRICULAR TIME INTERVALS IN NEWBORN INFANTS:  EVALUATION OF
TECHNIQUES FOR SURVEILLANCE OF FETUSES AT RISK FOR CONGENITAL
HEART BLOCK

Gunnar Bergman, L.-A. Jacobsson, M. Wahren-Herlenius* and S.-E. Sonesson

Department of Woman and Child Health, Pediatric Cardiology Unit, Astrid Lindgren Children‟s
Hospital, *Department of Medicine, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden


Objective
To evaluate one novel and two previously reported Doppler flow velocimetric techniques to
estimate atrioventricular (AV) time intervals, suggested to be useful for early identification of
fetuses at risk for congenital heart block.

Methods
In twenty-two newborn infants, Doppler tracings were obtained from the mitral valve/aortic outflow
and the superior vena cava/ascending aorta, simultaneously as an ECG was recorded. AV time
intervals were measured by using the onsets of the mitral A wave/aortic outflow (MV-Ao), superior
vena cava a wave/aortic flow (SVC-Ao), and mitral A wave/mitral valve closure (MV) as indirect
markers of electrical atrial and ventricular activation, respectively. AV time measurements were
repeated by a second observer to evaluate interobserver variability.

Results
Close positive linear relationships to the electrocardiographic PR interval were demonstrated for the
MV-Ao (r=0.82, Sy/x=7.4 ms), SVC-Ao (r=0.85, Sy/x=6.8 ms), and MV approach (r=0.92, Sy/x=3.8
ms). Both techniques using the aortic flow to indicate ventricular activation overestimated the PR
interval, the MV-Ao [+32±7.7 ms (mean±SD)] and the SVC-Ao approach (+22±7.0 ms). The new
MV approach using mitral closure for the same purpose did not overestimate the PR interval, but
there was a trend to underestimate the as An excellent agreement between observers was found for
all three methods.

Conclusions
When systematic differences between echocardiographic and electrocardiographic atrioventricular
time intervals are compensated for, all three techniques are useful to get indirect estimates of the PR
interval. As MV recordings only need insonation of a single valve, and accordingly are easier to
obtain, this technique fetuses in need for further surveillance. superior.




                                                    23             Developmental & Perinatal Biology 2006
                                                                                                  O9

CHARACTERIZATION OF THE UPA-MEDIATED CLEAVAGE OF PDGF-DD

Monika Ehnman and Ulf Eriksson

Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Stockholm,
Sweden


The Platelet-derived growth factor family consists of four members, which all exert their function
by forming disulfide-linked dimers. PDGF-A and PDGF-B, referred to as the classical members,
have been extensively studied for their physiological and pathological importance, partly by their
ability to recruit mural cells and thereby increase the stability of the vessel wall and prevent
endothelial hyperplasia, but also for their involvement in fibrotic disease and cancer.

In contrast to the classical PDGFs, the novel and closely related PDGF-C and PDGF-D need to be
extracellularly processed before they can mediate PDGF receptor activation. We have previously
identified tissue plasminogen activator (tPA) as an activator of PDGF-CC (1) and furthermore we
have characterized the direct protease-substrate interaction and the PDGF-C cleavage site (2).

Recently, urokinase (uPA) was identified as a proteolytic activator of PDGF-DD (3). This was
predicted since uPA, just like tPA, is a plasminogen activator accounting for the plasminogen
activation in the fibrinolytic system. In contrast to the more broad specific protease plasmin, which
is also able to process the novel PDGF members, the plasminogen activators have very few known
substrates indicating a more specific and fine-tuned regulation capacity in vivo.

Here we elucidate the uPA-mediated cleavage of PDGF-DD, show on a direct protein-protein
interaction and thoroughly characterize the potential cleavage site by alanine scan mutagenesis. Our
data support that PDGF-DD is activated by uPA in vivo and indicate that the cleavage efficacy can
be both negatively and positively modulated by amino acid substitutions in the R247-K257 region.
We are now investigating the importance of the urokinase plasminogen activator receptor (uPAR) in
the activation process of latent PDGF-DD.

   (1) Fredriksson L, et al. EMBO J (2004) 23, 3793-3802
   (2) Fredriksson L, et al. JBC (2005) 280, 26856-26862
   (3) Ustach C, et al. Mol Cell Biol (2005) 25, 6279-6288




                                                   24            Developmental & Perinatal Biology 2006
                                                                                                    010

IDENTIFICATION AND FUNCTIONAL VALIDATION OF GENES SELECTIVELY
EXPRESSED IN VASCULAR ENDOTHELIAL CELLS

Elisabet Wallgard1,2, Mats Hellström1,2, Liqun He1, Erik Larsson3, Mattias Kalén1,2, Minoru
Takemoto1, Noomi Asker2, Linda Karlsson2,4, Perry Hackett5, Per Lindahl3 and Christer Betsholtz1
1
  Karolinska Institutet, Stockholm, Sweden
2
  AngioGenetics AB, Göteborg, Sweden
3
  Wallenberg Laboratory of Cardiovascular Research, Sahlgrenska University Hospital, Göteborg,
Sweden
4
  Department of Physiology, Göteborg University, Göteborg, Sweden
5
  Discovery Genomics Inc., Minneapolis, Minnesota, USA


The aims of this ongoing study are to identify and characterize novel genes and proteins with
specific vascular expression by transcriptional profiling, in situ hybridization and functional studies
in zebrafish and mouse. Publicly available expression profiles are also used to identify candidate
genes.

We have identified a number of genes with endothelial cell-restricted expression using customized
spotted mouse cDNA microarrays. The cDNA library originates from RNA derived from isolated
vascular fragments/endothelial cells, resulting in a library enriched for the microvascular
transcriptome. Non-radioactive in situ hybridization was used to confirm an endothelial cell
restricted expression pattern for several of the candidates. Functional validation of 50 selected genes
was performed by gene knockdown in zebrafish using morpholino antisense technology.
Knockdown of 16 out of the 50 genes resulted in a vascular phenotype.

The public repositories of expression profiles are growing extensively, but expression data on
isolated vessels, in particular microvessels, is rare. Novartis has gathered a large number of
expression profiles from several tissues and cell types in the database GNF SymAtlas, which in this
study is combined with the vascular microarray data set and explored with bioinformatic tools, such
as clustering. Focus is put on the heterogeneity of endothelial cells and to characterize the
transcriptomes of different subsets of endothelial cells, e.g. quiescent-, angiogenic-, microvessel-
and large vessel endothelium.




                                                    25             Developmental & Perinatal Biology 2006
                                                                                                O11
STUDYING   PHASE  SINGULARITIES, ACTIVATION RATES (DOMINANT
FREQUENCIES), AND CONDUCTION BLOCK DURING HUMAN VENTRICULAR
FIBRILLATION

Nimalan Thavandiran1, K. Nanthakumar2, Stéphane Massé3, Rajesh Dhopeshwarkar2, Elias
Sevaptsidis4 and John Asta4
1
  Engineering Science, University of Toronto, Toronto, Canada
2
  Division of Cardiology, University Health Network, Toronto, Canada
3
  Biomedical Engineer, University Health Network, Toronto, Canada
4
  EP Technologist, University Health Network, Toronto, Canada


Sudden cardiac death is responsible for 40 000 deaths every year in Canada. Ventricular
Fibrillation is the leading cause of this mortality. The underlying mechanisms of Ventricular
Fibrillation in humans are not known. We studied explanted hearts from eight transplant recipients
undergoing a cardiac transplant. The human hearts were perfused with Krebs-Henseilet solution.
The epicardial and endocardial mapping was constructed with 112 electrodes predisposed on an
extensible sock and balloon array. The unique time course of an excitable element in cardiac tissue
can be represented as the phase of its trajectory in state space. The phase maps were constructed
using a time-delay embedding method. Activation rate and conduction block were estimated from
the Fast Fourier Transform. We describe global distribution phase singularities, dominant
frequency, and conduction block in human hearts. Results are to be presented.




                                                  26            Developmental & Perinatal Biology 2006
                                                                                                 O12
PROTEIN NITRATION CAUSES NEURONAL DEATH FOLLOWING TRAUMATIC
BRAIN INJURY

Michael Jones, Anthony Lau, Hong-Shuo Sun and Michael Tymianski

Department of Physiology, University of Toronto, Toronto, Canada; Division of Applied and
Interventional Research, Toronto Western Research Institute, Toronto, Canada


Background: Traumatic brain injury (TBI) afflicts approximately 1.4 million individuals and
presents a significant economic impact. The primary injury results in rapid neuronal death from
mechanical forces alone and is untreatable. Meanwhile, neurotoxic intracellular signaling pathways
are activated after the initial trauma. It is this secondary injury that offers potential for
neuroprotection by pharmacological means. Previously in our lab, it has been shown in an in vitro
model of secondary TBI, excess production of superoxide (O2-) reacted with nitric oxide (NO),
forming peroxynitrite (OONO-) which was lethal to neurons. Inhibition of NO production was
demonstrated to be neuroprotective. Subsequently, a whole animal model of TBI was shown to
result in increased 3-nitrotyrosine, a marker for peroxynitrite. What remains unclear is the
relationship between protein nitration and neuronal damage. Our main hypothesis is that
peroxynitrite causes neuronal death after TBI.

Methods: Using the lateral fluid percussion injury (FPI) model, male Sprague-Dawley rats were
given a moderate TBI (2.4 atm). Immunostaining for 3-nitrotyrosine, a marker for peroxynitrite,
was used in conjunction with NeuN, GFAP, and Fluoro-Jade to determine the relationship between
protein nitration and neuronal death. Western immunoblots were used to determine the time course
of protein nitration following FPI, and mass spectrometry was utilized for the identification of
nitrated proteins. GAPDH activity was also assessed following incubation with peroxynitrite.

Results: Preliminary results suggest that a temporal increase in protein nitration occurs in injured
neurons immediately following injury and lasting for at least 24 hours, however, protein nitration
does not occur in astrocytes. Injured neurons exhibit nitrated proteins in the cytoplasm and have
altered morphology compared to non-nitrated neurons. Furthermore, GAPDH was identified as a
nitrated protein and its activity was inhibited by peroxynitrite.

Conclusions: Our results suggest that, following FPI, peroxynitrite is rapidly formed, selectively
nitrating key glycolytic enzymes. In addition, it is believed that the formation of peroxynitrite is
closely related to neurodegeneration.




                                                   27            Developmental & Perinatal Biology 2006
                                                                                                 O13
EXPRESSIONAL DIFFERENCES IN ENDOGENOUS CONTROL GENES AND IN GENES
CONTROLLING MYELINATION, CELL DEATH/SURVIVAL AND SIGNALING IN
MOTOR AND FRONTAL CORTICES OF HUMAN CHRONIC ALCOHOLICS

Sofia Johansson1, Clive Harper2, Yasmin L. Hurd3, Georgy Bakalkin1 and Tomas Ekström1
1
  Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
2
  Discipline of Pathology, University of Sydney, NSW Australia
3
  Departments of Psychiatry and Pharmacology, Mount Sinai School of Medicine, New York, USA


Purpose: This study set out to investigate the stability of common endogenous control gene
expression for use as reference in quantification analyses of frontal and motor cortex from human
chronic alcoholics and controls. Furthermore, 45 genes regulating cell death/survival, myelination,
signaling and inflammation were quantified in 15 human alcoholics and 15 matched controls.

Methods: 1. The expression analysis of 16 commonly used internal control genes using total RNA
from frontal and motor cortex of chronic alcoholics and controls, were performed by using
TaqMan® Low Density Endogenous Control Panel (Applied Biosystems, ABI, Foster City, CA).
The expressional stability was analyzed using the Microsoft Excel based geNORM program freely
available for research purpose (http://medgen.ugent.be/genorm/). 2. We also applied micro fluid
Low Density Arrays (LDA, ABI) to evaluate the expressional differences in 45 selected genes.
Statistical analysis was used to determine possible group differences for each gene based on the
relative quantities calculated using the normalization factor determined from the geNORM stability
analysis.

Results: The most reliable genes for use as endogenous controls within the 16 analyzed turned out
to be different between the two brain regions investigated. Furthermore, the expression of three
control genes in motor cortex and one gene in frontal cortex were significantly different in
alcoholics as compared to controls. Moreover, genes regulating myelination, cell death/survival,
signaling and inflammation were differentially expressed in alcoholics as compared to controls,
many of which are regulated by NF-B.

Conclusions: The use of various real-time PCR methods with human tissues, require stringent
selection of endogenous controls in order not to get biased quantity data. This study shows that the
stability of endogenous controls can differ between brain regions, and also the expressional level
can differ between alcoholics and controls. Furthermore, the differences in gene expression in the
groups of genes analyzed imply a dysregulation of myelination and cell survival processes in
alcoholic cortices.




                                                   28            Developmental & Perinatal Biology 2006
                                                                                                  O14
SIRT1 DEACETYLASE-DEPENDENT AND   -INDEPENDENT EFFECTS OF
RESVERATROL AND RED WINE ON NEURAL STEM AND NEUROBLASTOMA
CELLS

Karolina Wallenborg1, P. Vlachos2, L. Huijbregts1, B. Joseph2 and O. Hermanson1
1
    CMB & Neuroscience, 2IMM, Karolinska Institute, Stockholm, Sweden


Histone deacetylases control chromatin modifications and gene expression. Of class III HDACs,
SIRs, only SIRT1 has been shown to be a nuclear deacetylase involved in transcriptional regulation
in euchromatin. To study potential roles for SIRT1 in neural differentiation, we performed studies
in FGF2-expanded cortical neural stem cells (NSC) from rodent embryos. The SIRT1 activator
resveratrol repressed neuronal differentiation of NSC significantly at low doses (1-2 µM) and this
effect could be completely reversed by SIRT1 siRNA. Resveratrol did not influence astrocytic
differentiation. Resveratrol-mediated activation of SIRT1 homologues has been associated with
increased longevity and red wine contains high levels of resveratrol. Surprisingly, slightly increased
levels of resveratrol (5-10 µM) induced increased cell death in NSC that was not fully reversed by
SIRT1 siRNA. We stimulated NSC with red wine with documented high resveratrol content and
compared to treatment with white wine devoid of resveratrol with the exact same ethanol content.
Red wine inhibited neurogenesis and induced a massive cell death. Importantly, neurogenesis,
astrocyte differentiation, and cell viability were unperturbed by treatment with white wine. In
human neuroblastoma cells, resveratrol and white wine had little or no effect whereas red wine
induced a rapid, necrotic cell death. Thus the effects of SIRT1, SIRT1 inhibition, resveratrol, and
red wine can be completely dissociated.




                                                    29            Developmental & Perinatal Biology 2006
                                                                                                 O15
MATERNAL ENDOWMENT OF BCL-2 FAMILY MEMBERS IN HUMAN AND MURINE
OOCYTES

Ingrid Lai1,2 and Andrea Jurisicova1,2
1
  Division of Reproductive Sciences, Department of Obstetrics and Gynecology and Department of
Physiology, University of Toronto, Toronto, Canada
2
  Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada


Background: In vitro fertilization (IVF) is an advanced technique employed to overcome
infertility. The outcome of IVF procedures is dependent on embryo quality, which is determined by
initial oocyte quality. In preimplantation embryo development, a large number of embryos become
arrested and/or fragmented. Existing literature points to apoptosis (programmed cell death) as a key
determiner of overall oocyte and embryo viability. In this regard, members of the Bcl-2 protein
family are implicated in oocyte and embryo viability as they are involved in the regulation of
apoptotic pathways. The Bcl-2 family consists of pro-apoptotic and anti-apoptotic proteins, and it is
thought that an overall imbalance of these proteins results in the decision to undergo or not undergo
apoptosis.

Aims: A preliminary screen of 14 human oocytes suggests that oocytes have a variable distribution
of Bcl-2 family members. In a parallel screen of oocytes from mice of advanced age, there was an
increased level of pro-apoptotic protein Bax. The current study investigates the expression levels of
several Bcl-2 family members, both pro- and anti-apoptotic, in human oocytes to establish whether
there are correlations between the distribution profile of these proteins and clinical parameters.

Methods: A screen of Bcl-2 family proteins will be performed on immature germinal vesicle,
immature MI, and unfertilized MII human oocytes obtained with consent from patients undergoing
hormonal stimulation. The expression levels of a subset of Bcl-2 family members in human oocytes
will be assessed using real time RT-PCR. From the findings of the screen, a subset of the Bcl-2
family proteins will be analyzed by immunocytochemistry.

Potential Outcomes: Based on the distribution profiles of Bcl-2 family proteins, we may determine
whether variability occurs within each patient and/or between different patients or cohorts. It is
expected that oocytes from females of advanced maternal age and oocytes from younger females
with compromised fertility will have an overall greater proportion of pro-apoptotic Bcl-2 family
members.




                                                   30            Developmental & Perinatal Biology 2006
                                                                                                  O16
PODOCYTE-SPECIFIC KNOCK-OUT OF MAMMALIAN TARGET OF RAPAMYCIN
(MTOR):   ITS ROLE IN GLOMERULAR DEVELOPMENT, FUNCTION AND
PATHOLOGY

Aarti Paltoo1,2 and Susan Quaggin1,2 ,3
1
  Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
2
  Department of Physiology, University of Toronto, Toronto, Canada
3
  St. Michael‟s Hospital, Toronto, Canada


Introduction
The glomerular filtration barrier plays an important role in maintaining the integrity of the kidney.
Functional or structural disruption of the glomerular filtration barrier leads to macromolecules
spilling into the urinary space. One of these macromolecules is albuminuria and it is the single
greatest predictor of progressive renal disease.

Several clinic papers have shown that a subset of patients treated with rapamcyin, a pharmacologic
inhibitor of mTOR (mammalian target of rapamcyin), develop marked proteinuria. The results
drawn from these papers have lead to the disposition that mTOR, specifically in the podocytes, is
required to regulate expression of VEGF-A; a key factor required for maintenance of the structure
and function of the glomerular filtration barrier.

Methods and Preliminary Results
Preliminary experiments were performed using wild type ICR mice treated with rapamycin once a
day for 2 weeks (gavage dosage- 2mg/kg/day, 5mg/kg/day & 10mg/kg/day). Kidneys from these
mice were harvested and prepared for H & E staining. Several developed proteinuria and focal
structural glomerular changes.

To further establish the relevance of mTOR in the glomerular filtration barrier, a genetic knock-out
of mTOR in podocytes is being made using the BAC recombineering and Cre-loxP system.

Preliminary Conclusions and Predicted Outcomes
Preliminary data obtained from wild type ICR mice treated with rapamycin supports the role of
mTOR in the glomerulus and also validates the usage of a mouse model.

Predicted outcomes for the mTOR podocyte specific-knockout is that it will lead to decreased
VEGF-A expression and subsequent massive proteinuria due to structural and functional breakdown
of the glomerular filtration barrier.




                                                   31             Developmental & Perinatal Biology 2006
                                                                                                    017
A GENETIC APPROACH FOR ESTABLISHING NOVEL STEM-CELL LINES FROM
LINEAGES OF POSTIMPLANTATION STAGE MOUSE EMBRYOS

Ridham Desai1, Gusztav Belteki2 and Andras Nagy1
1
    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
2
    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK


One of the major challenges of future medicine is to produce therapeutically useful progenitor cells
in large enough numbers. The process of in vitro differentiation of embryonic stem (ES) cells and
subsequent isolation of desired cell types is inefficient as well as time and cost intensive. The broad
aim of my graduate project is to develop a genetic strategy to derive permanent cell lines from cell
lineage progenitors of postimplantation stage embryos.

A vast number of different types of progenitor cells are present during prenatal development. These
progenitors go through multiple rounds of proliferation periods followed by differentiation steps
until they reach their final functional state. The progenitors require activation of specific signaling
pathways for the maintenance of their self-renewing properties.

Our working hypothesis is that when these progenitors are removed from their complex in vivo
differentiation environment, a finite number of specific signaling pathways or expression of
transcription factors will be sufficient for maintenance of their self-renewal. Finding these pathways
for each of these progenitors, followed by their regulated activation utilizing inducible genetic tools
would allow for their in vitro propagation. Such a manipulation could lead to the establishment of
permanent cell lines from these cells. Similar to ES cells, these cells will retain both their
developmental potential and commitments specific to the cell of origin. Such cell lines will be
called lineage committed stem cells or LCS cells.




                                                    32             Developmental & Perinatal Biology 2006
                                                                                                 O18

PGE2 CONCENTRATION AND COX-2 PROTEIN EXPRESSION IN HUMAN AMNION
EPITHELIAL AND MESENCHYMAL CELLS TREATED WITH MELOXICAM,
ARACHIDONIC ACID AND PROSTAGLANDIN E2

Valerija Rac1, I. Kamikovski1, John Challis2 and Stephen J. Lye1,2
1
    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
2
    Departments of Obs/Gyn and Physiology, University of Toronto, Toronto, Canada


Objective: Preterm birth occurs in 5-10 % of all pregnancies and is associated with considerable
neonatal mortality and morbidity. Effective and safe drugs to prevent preterm labour are not
currently available. The onset of parturition is caused by increased production of prostagladins due
to increased expression of inducible COX-2 isoform. In human pregnancy, the amnion is the site of
a major increase in COX-2 mRNA and ezyme activity. We used amnion primary cell culture system
to determine the effects of IL-1, arachidonic acid (AA), prostaglandin E2 (PGE2) and more
selective treatment selective cyclooxygenase-2 inhibitor meloxicam (MEL) on PGE2 concentration
and COX-2 enzyme protein expression.

Methods: Placenta and fetal membranes were obtained from patients at term at elective Cesarean
section prior to labour. Amnion was peeled from chorion and incubated with 0.25%Trypsin (amnion
epithelial cells - AE cells) or 0.1% Collagenase (amnion mesenchymal cells - AM cells). Cells were
plated on 6-well plates coated with collagen IV or fibronectin with appropriate media for each cell
line. Amnion epithelial and mesenchymal cells were stimulated with IL-1 (10ng/ml) and treated
with MEL (10M), AA (1 M) and PGE2 (10ng/ml) for 24 hours with appropriate controls.
Western blot analyses were used to compare COX-2 protein expression under the basal condition,
and with IL-1, MEL, AA and PGE2 treatment in both cell lines. Cell media was collected and
ELISA was performed to determine the level of PGE2. LDH cytotoxicity assay was performed to
determine cells viability.

Results: a) IL-1 stimulation of AE and AM cells resulted in increased PGE2 levels in media. b)
AA (1M) treatment resulted with significantly higher PGE2 levels in AE cells but had no effect on
AM cells. c) MEL (10M) treatment significantly inhibited the IL-1 stimulated PGE2 levels in AE
and AM cells. d) IL-1 stimulated COX-2 expression (w/out AA) in both AE and AM cells. e)
MEL treatment decreased COX-2 protein expression in IL-1 stimulated AE and AM cells. f) PGE2
(10ng/ml) treatment of AE and AM cells caused an increase in media PGE2 levels that was not
attenuated with MEL treatment.

Conclusion: This study shows that the potential tocolytic MEL can act to downregulate cytokine
induced PGE2 production in human amnion. This suggests that MEL may be useful in targeting
PGE2 production associated with preterm labour.




                                                   33            Developmental & Perinatal Biology 2006
                                                                                                  O19
HYPOTHALAMIC ADIPONECTIN RECEPTOR GENE EXPRESSION IS MODULATED
IN ADULT MOUSE OFFSPRING EXPOSED TO A MATERNAL DIET HIGH IN FAT AND
PROTEIN DURING PREGNANCY AND LACTATION

Dyan Sellayah, Kimberley D. Bruce, Chunli Wang, Frederick W. Anthony, Mark A. Hanson,
Christopher D. Byrne and Felino R.A. Cagampang

Centre for Developmental Origins of Health and Disease, University of Southampton, Princess
Anne Hospital (F-887), Southampton, UK


Introduction: Adiponectin, an adipokine found exclusively in adipose tissue, has been shown to
exert insulin-sensitising effects via activation of adiponectin receptors, namely Adiponectin
Receptor 1 (AdipoR1) and Adiponectin Receptor 2 (AdipoR2). AdipoR1 is abundantly expressed in
skeletal muscle whereas AdipoR2 is mostly found in the liver. However, little is known about the
expression pattern of these receptors in the brain, although adiponectin serum-cerebrospinal fluid
transport has been identified. The aims of this study were to characterise AdipoR1 and AdipoR2
gene expression in the mouse hypothalamus, and determine whether exposure to maternal high fat-
high protein diet (HFP) in-utero and during lactation modulates expression levels in adult offspring.

Methods: Female Balb/C mice were either fed a high fat-high protein diet (36.8% CHO, 32%
lipid, 28% protein; n=9) or standard chow (68.8% CHO, 10% lipid, 18% protein; n=6) six weeks
prior to conception through to pregnancy and lactation. Weaned offspring were fed standard chow
until adulthood. Offspring were killed at 8 weeks old and hypothalamic brain blocks collected and
analysed for changes in gene transcript levels by MT-qcPCR.

Results: In all offspring, hypothalamic AdipoR1 mRNA levels were 4-fold higher compared with
AdipoR2 (p<0.001). Moreover, AdipoR1 and AdipoR2 levels in males were 71% (p<0.05) and
148% (p<0.001) higher respectively at baseline (offspring of chow-fed mothers) than in females. In
HFP male offspring, but not in females, there was a 16% reduction (p0.05) in hypothalamic
AdipoR2 mRNA levels compared to those from mothers fed the chow diet. There was no significant
difference in AdipoR2 levels between the female chow and HFP groups. There was also a trend
towards a reduction in AdipoR1 levels in both HFP male and female offspring but this was not
found to be significant.

Conclusions: We have found that, not only are the receptors for adiponectin expressed in the
hypothalamus, but also the level of gene expression for AdipoR1 is much higher than AdipoR2. We
have also shown that there are sex specific differences in the level of expression for these receptors
in this part of the brain. Furthermore, we have observed that AdipoR2 expression is modulated in
males, but not in female offspring from mothers fed a HFP diet during pregnancy and lactation. In
light of recent studies suggesting a role for adiponectin in hypothalamic regulation of energy
expenditure, our results may be part of the developmental changes in energy expenditure regulation
that have sex-specific consequences in later life.




                                                    34            Developmental & Perinatal Biology 2006
                                                                                                O20
CHRONIC MATERNAL ADVERSITY AND OFFSPRING GROWTH, BEHAVIOUR AND
HYPOTHALAMO-PITUITARY-ADRENAL (HPA) FUNCTION: MALE
VULNERABILITY

Jeff Emack1, Alice Kostaki1, Dominique Walker4 and Stephen Matthews1,2,3
1
  Departments of Physiology, 2Obstetrics and Gynecology, and 3Medicine, Faculty of Medicine,
University of Toronto, Toronto, Canada
4
  Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal,
Québec, Canada


Prenatal stress has been linked to an increased risk for impaired behavioural and emotional
development and affective disorders in humans. Further, acute periods of prenatal stress have been
shown to have profound effects on HPA function in animal models. However, humans are more
likely to experience chronic stress during pregnancy rather then acute periods. Therefore, the
objective of this study was to determine the effects of chronic maternal adversity (MA) during the
2nd half of pregnancy and early postpartum period on growth, behaviour and HPA axis function in
guinea pig offspring. Methods: For the MA paradigm, pregnant guinea pigs were exposed to a
stressor every 2nd day for the 2nd half of gestation (gestational day (GD) 32 to 66) and from
postnatal day (PND) 1 until weaning on PND 25. The stressors were forced foraging (food
distributed throughout bedding, 24h), food restriction (10h), novel environment (2h) and social
stress (unfamiliar female, 2h). A group of control animals remained undisturbed throughout
pregnancy and the perinatal period. Weight and salivary cortisol were measured in mothers during
pregnancy and animals were allowed to deliver normally. In the offspring, weight was measured at
birth and every 5 days and basal salivary cortisol was measured at PND 15. On the day of weaning
(PND 25), guinea pig offspring underwent an open field test to assess locomotor activity and
anxiety and salivary cortisol samples were taken to assess activated HPA axis function. Results:
MA mothers displayed increased salivary cortisol levels in the later stages of pregnancy compared
to control mothers (P < 0.001). The male offspring of MA mothers tended to weigh less at birth
than controls and this lower weight was maintained up to weaning (P < 0.001). There was no effect
in female offspring. In open field testing, MA male offspring showed a decrease in total activity
compared to controls (P < 0.05), and the animals spent more time in the corners of the open field,
indicating increased anxiety. MA male offspring also displayed a blunted HPA axis response to the
novel environment of the open field. Again, there was no effect of MA on behaviour in female
offspring. Conclusion: Chronic maternal adversity leads to modification of growth trajectory,
anxiety behaviour and HPA response to stress. Further, male offspring are more vulnerable than
females to the effects of chronic maternal adversity during the perinatal period.




                                                 35             Developmental & Perinatal Biology 2006
                                      ABSTRACTS

                                         - Posters -
P1
Valeria DiGiovanni and Norman Rosenblum. The role of BMP signalling in renal development.

P2
Enrica Fornaro and Jacques Belik.         SSRI‟S use during pregnancy and fetal pulmonary
hypertension.

P3
Alevtina Galtseva, Lynne Quarmby and Norman Rosenblum. Rose of cilia in sonic hedgehog
signaling during kidney development.

P4
Paul Gill and Norman Rosenblum. Characterization of enhanced hedgehog signaling on renal
development.

P5
Lisa Hollis, F.W. Anthony, M.A. Hanson and L.R. Green. Hepatic glucocorticoid receptor (GR)
expression following early gestational and/or postnatal undernutrition in adult sheep.

P6
Venu Jain, Lee Adamson and John Kingdom. Induction and characterization of IUGR by sub-
lethal injections of Gcm-1 siRNA into the embryonic exocoelomic cavity in mice.

P7
Kusala Jayasuriya and Melanie Woodin. Developmental regulation of GABA and KCC2 in the
Zebrafish (Danio rerio) central nervous system.

P8
Subhash Juneja, Ameet Sengar, Kelvin Wang, Brenda Cohen, Connie Chen and Sean Egan.
Intersectin-1 and –2 double mutant mice show multiple defects.

P9
Amita Kapoor and Stephen G. Matthews. Prenatal stress modifies endocrine function and
behaviour in female guinea pig offspring: Interaction with the reproductive cycle.

P10
Maya Nisancioglu and Christer Betsholtz. Generation and initial characterization of mice lacking
pericyte gene RGS5.

P11
Linus Olson, Osuke Iwata, Carina Lothian, Viktoria Martin, Fredrik Setterwall, Nicola J. Robertson
and Hugo Lagercrantz. Hypothermia with a PCM madress as treatment of neonatal encephalopati.

                                                  36            Developmental & Perinatal Biology 2006
P12
Magnus Sandberg and Jonas Muhr. Identification of cis elements regulating Sox21 expression.

P13
Erik Södersten and Ola Hermanson. The zinc finger/BTB domain-containing protein RP58 is a
BMP-regulated transcriptional repressor in neural development.

P14
Johanna Sundqvist, Linda Csöregh, Eva Andersson and Gabriel Fried. Subcellular localization of
-, - and -secretases in primary cell cultures of human neurons and glia cells.

P15
Clare Tower, John Aplin and Philip Baker. The role of angiotensin II receptors at the maternal-
fetal interface and their interaction with transforming growth factor .

P16
Jakob Carlsson, Marco Bartocci, Matteo Bruschettini and Hugo Lagercrantz. Activation of
temporal cortex in 8-month old infants after visual exposure to their mother‟s face and to an
unknown face. A near infrared spectroscopy study.




                                                37            Developmental & Perinatal Biology 2006
                                                                                                   P1
THE ROLE OF BMP SIGNALLING IN RENAL DEVELOPMENT

Valeria Di Giovanni and Norman D. Rosenblum

The Hospital for Sick Children, Toronto, Canada; Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Canada


Renal dysplasia, defined as malformation of the tissue elements, is the major cause of childhood
renal failure. The molecular etiology underlying renal dysplasia is mostly unknown, however new
information about the molecular control of normal renal development has provided a conceptual
platform to examine possible pathogenic mechanisms. The kidney develops from reciprocal
interactions between two tissues derived from the intermediate mesoderm: the ureteric bud and the
metanephric mesenchyme. The collecting duct system of the kidney develops due to influence of
multiple growth factors in a process termed renal branching morphogenesis. Members of the bone
morphogenetic protein (BMP) family have been demonstrated to have an inhibitory role in renal
branching morphogenesis. Certain BMP proteins signal through a receptor complex consisting of
BMP receptor II and ALK3. The activated ALK receptor transmits the BMP signal inside the cell
via phosphorylation of the specific receptor associated protein, Smad1. Embryonic expression of
ALK3 has been demonstrated in both the ureteric bud and metanephric mesenchyme cell lineages.
Mice were created with a conditional deletion of ALK3 in these tissues to explore the role of ALK3
signalling during renal development. Conditional deletion of ALK3 in the metanephric
mesenchyme cell lineage demonstrated a possible defect in renal branching morphogenesis and a
delay in nephrogenesis. A mouse model of conditional deletion of ALK3 in the ureteric bud lineage
demonstrated an adult phenotype of increased cell proliferation, shrinkage of the inner medulla,
absence of the renal papilla and cortical cysts. As well, these mice also demonstrated decreased
protein levels of phosphorylated Smad1, which correlated with a decrease in beta-catenin protein
levels. As transgenic mice that express a constitutively active form of ALK3 in the ureteric bud
lineage develop cystic renal dysplasia and increased beta-catenin expression in the nuclei of cystic
epithelia, it has been hypothesized that ALK3 signalling may modulate beta-catenin protein levels
during renal development. Further analysis of possible beta-catenin regulatory mechanisms revealed
a decrease in protein levels of the inactive form of glycogen synthase kinase in mice with a deletion
of ALK3 in the ureteric bud lineage. As well, these mice revealed a decrease in expression of Pax2,
a marker of the induced metanephric mesenchyme. Bioinformatic analysis identified Smad binding
sites in the Pax2 promoter, pointing to the possible requirement of Smad signalling for Pax2
transcription.




                                                   38            Developmental & Perinatal Biology 2006
                                                                                                  P2
SSRI’S USE DURING PREGNANCY AND FETAL PULMONARY HYPERTENSION

Enrica Fornaro and Jacques Belik

Department of Pediatrics, The Hospital for Sick Children, Toronto, Canada


Depression is a common disease in women lifetime (10-25%) and pregnancy is a stressful state that
may aggravate depression and increase the need for medical therapy. SSRIs (fluoxetine and
paroxetine) are the standard form of pharmacotherapy for depression.

Recently an association between maternal use of SSRIs and persistent pulmonary hypertension of
the newborn (PPHN) was suggested.

SSRIs are not approved for use in human pregnancy. Despite this they are in common use for the
treatment of depression and other affective disorder during gestation. However there is limited
information on their pharmacokinetics and their interaction with lung development in fetus. We
propose to treat pregnant rats with SSRIs to verify if they induce PPHN.

SSRIs act inhibiting the serotonin transporters and it seems that after SSRIs administration plasma
serotonin increases. Serotonin is known as a vasoconstrictor that promotes pulmonary smooth-
muscle cell hypertrophy and hyperplasia. In adult the SSRIs were suggested as protector against
hypoxia PH.

Consequently, we also propose to induce pulmonary hypertension in the fetuses and in the
newborns and to administrate SSRIs to the dams or to the newborns to verify if they act as
protectors.

We would like to also analyze pulmonary artery smooth muscle cell putting them in culture fields
and seeing their proliferation in response to different supposed growth factors.

These experiments will contribute to better evaluate SSRIs safety in pregnant women.




                                                  39            Developmental & Perinatal Biology 2006
                                                                                                   P3
ROLE OF CILIA               IN   SONIC     HEDGEHOG         SIGNALING        DURING         KIDNEY
DEVELOPMENT

Alevtina Galtseva1,2, Lynne Quarmby1 and Norman Rosenblum1
1
    Department of Nephrology, The Hospital for Sick Children, Toronto, Canada
2
    Department of Physiology, University of Toronto, Toronto, Canada


Sonic Hedgehog signaling (Shh) plays critical roles in renal morphogenesis by controlling
expression of renal patterning genes and cell cycle modulators. Mice deficient in Shh in ureteric
bud lineage and mice deficient in truncated form of Gli3, one of the downstream effectors of Shh
pathway, display presence of renal hypoplasia or dysplasia. Recent reports that primary cilia,
microtubule-based organelles that extend from plasma membrane, are required for Shh signaling in
the embryonic node and during limb development provides the basis for our hypothesis that the
primary cilium plays essential roles during kidney morphogenesis. First, we determined the
temporal and spatial expression of cilia (defined by indirect immuno-fluorescence as acetylated-
tubulin positive rod-like structures) and co-stained with developmental markers. As early as E11.5
we observed cilia on the luminal surface of ureteric bud cells and on all metanephric mesenchyme
cells. By E13.5, populations of metanephric-derived cells demonstrated differential spatial
expression of cilia. In contrast to NCAM-positive cells, WT1-positive cells expressed cilia on their
presumptive apical surfaces. Beginning at approximately E15.5, luminal cilia lengthened. We
conclude that cilia are expressed in mesenchymal and epithelial kidney lineages during
embryogenesis. Electron microscopy is being used to further characterize cilia morphology. By
infecting cultured kidney explants with adenoviruses carrying Gli1-GFP, Gli2-GFP and Gli3-GFP
genes I will determine changes in subcellular localization of these downstream components of the
Hedgehog pathway in response to activated and blocked pathway. Based on observations in the
node and limb bud, I expect Gli‟s to localize to cilia when the signaling pathway is on. This
compartmentalization of signaling components might be the main determinant of their activity. To
better understand the significance of cilia in kidney morphogenesis a set of in situ hybridization
experiments will be performed to demonstrate expression pattern of Hedgehog signaling effectors in
a normal developing kidney. This will serve as groundwork for later analysis of kidney
development in presence of a ciliary mutation. Taken together the results that these experiments will
provide will reveal details on the mechanism by which cilia regulate kidney development.




                                                   40            Developmental & Perinatal Biology 2006
                                                                                                   P4

CHARACTERIZATION OF ENHANCED HEDGEHOG SIGNALING ON RENAL
DEVELOPMENT

Paul S. Gill and Norman D. Rosenblum

The Hospital for Sick Children, Toronto, Canada; Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Canada


During renal development, the epithelial ureteric bud induces the metanephric mesenchyme to
proliferate and differentiate into the functional units of the kidney. In reciprocal fashion, the
metanephric mesenchyme signals the ureteric bud to elongate and branch. Alterations in signaling
between these two cell lineages often results in malforming renal tissue elements. This disease,
termed renal dysplasia, is the major cause of childhood renal failure.

Sonic hedgehog (SHH) signaling plays a key role in mediating the development of many organs
during embryogenesis. Interestingly, Sonic hedgehog null mice, Shh-/-, display malformed kidneys
(renal dysplasia) implicating the signaling pathway in renal development. To better understand the
function of SHH signaling in renal development, Suppressor of Fused (SUFU), a downstream
signaling component, was deleted from the ureteric bud. Since SUFU had previously been
demonstrated to regulate GLI protein processing, promoting accumulation of processed GLI3 and
repressing accumulation of full length GLI1 and GLI2, we expected mice with deletion of SuFu
from the ureteric bud to serve as models of enhanced SHH signaling in the ureteric bud cell lineage.
Here we begin to characterize the renal phenotype of SuFu-/loxP; HoxB7 Cre mice.




                                                   41            Developmental & Perinatal Biology 2006
                                                                                                       P5

HEPATIC GLUCOCORTICOID RECEPTOR (GR) EXPRESSION FOLLOWING EARLY
GESTATIONAL AND/OR POSTNATAL UNDERNUTRITION IN ADULT SHEEP

Lisa J. Hollis, F.W. Anthony, M.A. Hanson and L.R. Green

Centre for Developmental Origins of Health and Disease, University of Southampton,
Southampton, UK


Background: In human and animal studies environmental constraints during pre and post natal life
result in phenotypic changes that can be associated with increased cardiovascular and metabolic
disease risk in later life (Barker et al., 2002, Gardner et al., 2005, Poore et al., 2006). The liver is a
key organ in glucose and lipid metabolism. During hypoglycaemia in fetal sheep blood is
redirected from the liver in favour of the adrenal (Burrage et al., 2006). In humans, blood
redistribution via the ductus venosus creates a “liver sparing” effect in response to unbalanced diet
and lower maternal fat stores (Haugen et al., 2005). Maternal gestational low protein diet in rats
alters adult hepatic gene expression (Lillycrop et al., 2005) with distinct effects between liver lobes
(Zhang & Byrne, 2000). We are investigating the effect of reduced early gestation maternal
nutrition and/or early post natal life undernutrition on the expression of the glucocorticoid receptor
(GR) in adult sheep liver. This gene is of particular interest because perturbations in its expression
are associated with disturbances in cardiovascular and metabolic control.

Methods: Welsh Mountain ewes received 100% (C, n=36) or 50% of total nutrient requirements
(U, n=39) from 1-31 days of gestation, and 100 % thereafter. Offspring were fed ad libitum (CC,
n=20; UC, n=19) or to reduce body weight to 85% of individual target weight from 12 to 25 weeks
postnatal age and ad libitum thereafter (CU, n=17; UU, n=21). Each group contained approximately
equal numbers of males and females and the ratio of twins to singletons was ~2:1. Offspring were
sacrificed at 2.5 years of age, the livers were harvested and segments from the left and right liver
lobes were frozen in liquid nitrogen. GR mRNA levels were measured by semi quantitative RT-
PCR and will be corrected using a housekeeping gene. All data will be analysed by ANOVA.

Results: Preliminary results (uncorrected for housekeeping gene) in males indicate that early life
nutrition did not affect GR mRNA levels in left liver but GR expression was lower in twins
compared to singles.

Future work: Ongoing work includes measuring GR mRNA levels expression in male right liver
and in female livers. Subsequent work may involve investigation of possible epigenetic
mechanisms linking early life environment and post natal phenotype e.g. gene methylation studies.
Supported by the BBSRC




                                                      42             Developmental & Perinatal Biology 2006
                                                                                                   P6

INDUCTION AND CHARACTERIZATION OF IUGR BY SUB-LETHAL INJECTIONS OF
GCM-1 SIRNA INTO THE EMBRYONIC EXOCOELOMIC CAVITY IN MICE

Venu Jain1,2, Lee Adamson1,2 and John Kingdom1,2
1
    Obstetrics & Gynecology / Maternal-Fetal Medicine, Mount Sinai Hospital, Toronto, Canada
2
    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada


The transition from an implanted embryo to a 3.5 kg term baby is dependent upon the attainment of
normal placental function. Key tasks for the placenta to achieve this goal are mediating maternal
cardiovascular adaptation to pregnancy and facilitating an adequate supply of oxygen and nutrients
for growth. Abnormal placental development in early pregnancy initiates the more severe
manifestations of two common diseases of pregnancy, namely pre-eclampsia and intrauterine
growth restriction (IUGR). These result in adverse clinical outcomes for the mother and fetus. In the
past decade, great advances have been made in understanding of how the various placental
components form a functional organ, and how we can utilize such knowledge to recognize abnormal
placental development before maternal and/or fetal complications have occurred. Nevertheless, our
understanding of the mechanisms leading to placental failure is rudimentary, and effective treatment
strategies are non-existent. Progress is being made using human placental explants and cell lines by
our group and others, but a suitable animal model to complement the human work would accelerate
our understanding and provide models to rapidly test new treatments and assess long term
outcomes. Genetically altered mice have rapidly advanced our knowledge of the genes and gene-
products critical for normal placental development. Our group has pioneered the development of a
state-of-the-art high resolution ultrasound system, the ultrasound biomicroscope, and have shown
that images and Doppler recordings, that are as good or better than those obtained in human
pregnancy, can now be obtained non-invasively from living mouse embryos throughout
development. The proposed research will address the hypothesis that defective chorio-allantoic
placental development can be created using ultrasound guidance to inject short inhibitory RNA
(siRNA) to glial cell missing-1 (Gcm-1) into the exocoelomic cavity, an embryonic cavity adjacent
to the developing placenta in the mouse. We further hypothesize that this intervention will result in
embryonic lethality and/or IUGR. These studies will attempt to induce and characterize IUGR due
to chorio-allantoic maldevelopment following sub-lethal injections of Gcm-1 siRNA injections into
the exocoelomic cavity, thereby validating a novel approach for studying the origins of placentally-
mediated IUGR.




                                                   43            Developmental & Perinatal Biology 2006
                                                                                                     P7

DEVELOPMENTAL REGULATION OF GABA AND KCC2 IN THE ZEBRAFISH (DANIO
RERIO) CENTRAL NERVOUS SYSTEM

Kusala Jayasuriya and Melanie Woodin

The Physiology Group, Department of Zoology, University of Toronto, Toronto, Canada


The neurotransmitter -amino butyric acid (GABA) has been shown to stimulate several essential
developmental functions, including neuronal migration, cell division, and neuronal growth. GABA
undergoes a dramatic developmental switch; it is excitatory in immature organisms while it is
inhibitory in the adult. The long-term objective of our study is to determine whether activity (i.e.
visual input) can regulate the time in development when this switch occurs. We have chosen the
visual system of zebrafish (Danio rerio) as our model system for its well-characterized advantages
as a molecular model system and its well-defined visual anatomy. As the first step in addressing our
long-term goal, we investigated the developmental expression of GABA in the retina. Zebrafish
embryos at 3-9 days post fertilization (pfd) were fixed and sectioned across the dorsal-ventral axis.
GABA expression was detected by immunocytochemistry followed by confocal microscopy.

Our results showed a transient expression of GABA in the retinal ganglion cell layer and the tectum,
in which the dispersed GABA expression observed at 3 pfd diminished and became more localized
to retinal layers and to the lobes of the tectum by 6 pfd. This is a critical period in development of
the CNS during which visual neural networks are being established. Thus, we suggest that GABA,
in addition to its functions as a classical neurotransmitter, may play a role in the development of the
visual system.

The switch in the polarity of the GABAergic response is determined by the expression of KCC2, a
neuron-specific K+/Cl- cotransporter. KCC2 rapidly extrudes chloride from developing neurons,
switching GABAergic transmission from excitatory to inhibitory. Using Western blot analysis, we
demonstrated that the neuron-specific expression of KCC2 starts at around 3 pfd, becomes
prominent at 6 pfd, and increases gradually until the adult stage. Therefore, the developmental
expression of GABA correlates with the up-regulation of KCC2. These results are the basis for
future studies to examine whether sensory-input can regulate the time at which the switch in
GABAergic synaptic transmission occurs via the regulation of KCC2 expression and to investigate
the role of GABAergic transmission in visually guided behaviours.




                                                    44             Developmental & Perinatal Biology 2006
                                                                                                    P8
INTERSECTIN-1 AND -2 DOUBLE MUTANT MICE SHOW MULTIPLE DEFECTS

Subhash Juneja, Ameet Sengar, Kelvin Wang, Brenda Cohen, Connie Chen and Sean Egan

Developmental Biology Program, Hospital for Sick Children, Toronto, Canada


Intersectin proteins (Itsn1 and Itsn2, also known as Ese1 and Ese2) bind Epsins, Eps15 as well as
Dynamin, and are known for their role in endocytosis. Our lab has generated -Geo-fusion knockin
(Itsn1Geo/Geo) and Itsn2 knockout (Itsn2exon3/exon3) mice. Single homozygous mutants for either
Itsn1 or Itsn2 are viable and fertile. Here we report that double homozygous mutants
(Itsn1Geo/Geo/Itsn2exon3/exon3) show multiple defects. Double mutant mice were produced by
intercrossing Het/mut(Itsn1Geo/+/Itsn2exon3/exon3) mice. At birth, their body weight was similar to
that of their littermates. However, their rate of increase in body weight was lower, and more than
65% pups died as runts before the age of 4 wks. Those pups which lived up to 4 wks, survived to
adulthood. Pups that died postnatally had an intestinal block at the ileum-cecum junction. The cause
of death may be related to a malfunction of cecum. Of the animals that survived to adulthood, the
majority of males were infertile. Most of males failed to mate females. The sperm showed tail kinks
and folds. Endocytosis and lysosome formation were reduced in non-ciliated luminal cells of vas
efferens. A model to explain male infertility in double mutants will be presented. In contrast, double
mutant females had reduced fertility but many gave birth. Following parturition, females were
unable to nurse their pups despite the presence of milk within mammary ducts. Mice showed
defects in pituitary morphology. Finally, most mice developed eye infections at some point between
6-12 months of age. These infections were irreversible, and did not respond to treatment. The
average lifespan of double mutant mice was, significantly lower than wildtype mice. Towards the
end of life, mice showed walking problem with hunching back and low level tremors. In summary,
Itsn1 and 2 double mutant mice showed early postnatal low growth rate, postnatal lethality, cecal
defects, male infertility, male mating defects, sperm defects, female nursing defects, pituitary
defects, eye infections, walking problems, tremors and a relative short lifespan. The potential
endocytic basis of these phenotypes is being studied to understand the role of intersectins in each
process.




                                                    45            Developmental & Perinatal Biology 2006
                                                                                                  P9
PRENATAL STRESS MODIFIES ENDOCRINE FUNCTION AND BEHAVIOUR IN
FEMALE GUINEA PIG OFFSPRING: INTERACTION WITH THE REPRODUCTIVE
CYCLE

Amita Kapoor1 and Stephen G. Matthews2
1,2
 Departments of Physiology, 2Obstetrics and Gynaecology and 2Medicine, Faculty of Medicine,
University of Toronto, Toronto, Canada


Background: Prenatal stress (PS) is associated with an increased risk of adult-onset pathologies.
We hypothesized that PS will affect hypothalamic-pituitary-adrenal (HPA) axis function and
behaviour in adult female guinea pig offspring however, these effects will change as a function of
reproductive cycle status and timing of the stress exposure.

Methods: Pregnant guinea pigs were exposed to a strobe light stressor on gestational days 50-52
(PS50) or 60-62 (PS60). In adulthood, ambulatory activity in an open field was assessed. Basal
and activated adrenocortical activity was evaluated by saliva sampling. For all testing, females
were divided on the basis of their reproductive cycle.

Results: During estrous, ambulatory activity was decreased in PS60 offspring (p<0.01) and the
free cortisol response to activation of the HPA axis was decreased in both PS50 and PS60 (p<0.01).
PS60 female offspring also exhibited lower plasma estradiol levels (p<0.001) and a smaller ovarian
to body weight ratio (p<0.05).

Conclusions: PS during critical windows of development programs HPA axis function and
behaviour in female adult offspring. Further, these changes appear to be reproductive cycle
dependent.




                                                  46            Developmental & Perinatal Biology 2006
                                                                                                 P10

GENERATION AND INITIAL CHARACTERIZATION OF MICE LACKING PERICYTE
GENE RGS5

Maya H. Nisancioglu and Christer Betsholtz

Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska
Institutet, Stockholm, Sweden


All normal blood microvessels harbour cells referred to as pericytes, which contribute to
microvessel formation and stability. The molecular mechanisms involved in these pericyte-mediated
functions are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of
microvascular abnormalities associated with diabetes and angiogenesis in tumours and this
highlights the need for a better understanding of pericyte signalling and their physiological
functions.

We initiated a microarray approach to detect genes differentially expressed in pericyte-deficient
mouse mutants, and identified Regulator of G-protein Signaling-5 (RGS5) as a novel pericyte gene
(Bondjers et al. Am J Pathol. 2003 Mar;162(3):721-9). RGS5 is a member of the RGS superfamily
and acts as a negative regulator of heterotrimeric G protein-mediated signalling through G protein-
coupled receptors. Interestingly, RGS5 is induced in pericytes during the “angiogenic switch” in
tumor angiogenesis and wound healing, suggesting that pericytes and RGS5 have a role in vascular
remodelling.

To study the role of RGS5 in vivo, we have generated a mouse line lacking the RGS5 gene. Our
targeting construct has replaced the RGS5 coding sequence with that of a GFP gene, allowing the
detection of pericytes using GFP as a reporter. Mice homozygous for the inactivated RGS5 are born
at an expected frequency of 25%, and their vasculature seems to develop normally. The detailed
phenotypical analysis of this mouse line is in progress. The generation of an RGS5 deficient mouse
line with GFP-positive pericytes gives us a tool to study the role of RGS5 and pericytes in vivo and
also provides a possibility for visualization and isolation of these vascular mural cells.




                                                   47            Developmental & Perinatal Biology 2006
                                                                                                  P11
HYPOTHERMIA WITH A PCM MADRESS AS TREATMENT OF NEONATAL
ENCEPHALOPATI

Linus Olson1,3, Osuke Iwata2, Carina Lothian1,4, Viktoria Martin3, Fredrik Setterwall3, Nicola J.
Robertson2 and Hugo Lagercrantz1
1
  Karolinska Institute, Department of Woman and child health, Neonatology Unit, Stockholm,
Sweden
2
  Department of Obstetrics and Gynaecology, University College London, Great Britain
3
  KTH, Royal Institute of Technology, Chemical Engineering and Technology, Division of Energy
Processes, Stockholm, Sweden
4
  Stockholm Söder Hospital, Neonatal Unit, Stockholm, Sweden


Recent results from three randomized clinical trials of mild hypothermia for newborn infants with
neonatal encephalopathy suggest that a useful therapy for neonatal hypoxic-ischaemic
encephalopathy is a real possibility. These and other on-going trials use methods to induce cooling
which require electricity and are relatively expensive. A “low tech” method of providing effective
and safe cooling to a precise body temperature is required for hypothermia to be applied to both the
developing as well as the developed world.

The importance of a “low tech” method of safely cooling infants with perinatal hypoxia-ischaemia
The standard method of cooling in the on-going international whole body cooling study in the
developed world (TOBY trial; is the Tecotherm mattress which needs a power supply, consumables
(a regular supply of coolant) and costs around £5,000). We are proposing to pilot a “low tech”
method that can effectively cool and maintaining the core temperature of an infant at a constant
level. Phase changing materials (PCMs) appear to be ideal for such a purpose. PCMs do not require
electricity, they are biologically safe for humans, are cheap, can be reused and are likely to provide
a more stable cooling temperature than other “low tech” methods such as ice packs.

We will assess and optimise a novel “low tech” cooling method using phase changing material
(PCM). This material requires no power or maintenance to be effective. It is envisaged that these
pilot studies using a piglet experimental model may be applied to newborn infants in the developing
world where clinical trials for mild cooling are urgently needed.




                                                    48            Developmental & Perinatal Biology 2006
                                                                                                P12
IDENTIFICATION OF CIS ELEMENTS REGULATING SOX21 EXPRESSION

Magnus Sandberg and Jonas Muhr

Ludwig Institute for Cancer Research, Karolinska Institutet, Stockholm, Sweden


The generation of neurons constitutes the foundation of nervous system development, yet the
mechanisms underlying neurogenesis are not well established. The HMG-box transcription factors
Sox1-3 have been shown to block the progression of neurogenesis and maintain neural cells in an
undifferentiated state. Another HMG-box protein, Sox21, has the opposite activity compared with
Sox1-3 and promotes neuronal differentiation. Sox21 appears to bind similar target sequences as
Sox1-3, but repress rather than activate gene transcription. These studies have revealed that the
balance of Sox1-3 and Sox21 activities determine whether neural cells remain as precursor cells or
commit to a neuronal differentiation program. To better understand how the activity level of Sox21
is controlled and gain insights into how precursor cells commit to neurogenesis, the aim of this
project is to elucidate how the expression of Sox21 is regulated at the molecular level. We have
started to identify highly conserved noncoding elements (HCNE) surrounding the Sox21 open
reading frame (ORF). These HCNEs might serve as potential cis-regulatory elements necessary for
correct Sox21 transcription. Three HCNEs, 3´of the Sox21 ORF were identified using a web based
program termed, ECR browser. One of the regions, 644 bases long, could direct the expression of a
reporter gene in a pattern resembling the endogenous Sox21 expression within the developing spinal
cord. Using a combined strategy involving mutagenesis, ChIP and cell transfection assays we hope
to generate a clear model of how the expression of Sox21 is regulated at the molecular level.




                                                 49             Developmental & Perinatal Biology 2006
                                                                                              P13

THE ZINC FINGER/BTB DOMAIN-CONTAINING PROTEIN RP58 IS A BMP-
REGULATED TRANSCRIPTIONAL REPRESSOR IN NEURAL DEVELOPMENT

Erik Södersten and Ola Hermanson

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden


The zinc finger/BTB domain-containing transcriptional repressor RP58 (ZFP238) has been shown
to interact with the co-repressor Dnmt3a in association with transcriptionally silent
heterochromatin. To elucidate a possible role for RP58 in forebrain development, we performed in
situ hybridization experiments on sections from mice of various embryonic ages and found that
RP58 mRNA expression was restricted to the dorsal regions of the telencephalon, partially
overlapping with expression of BMP factors. RP58 mRNA was detected in FGF-expanded cortical
neural stem cells (NSC) as assayed by real-time PCR. Consistent with the expression pattern in
vivo, RP58 gene expression was significantly up-regulated in NSC by 24 hours of BMP4 or BMP7
treatment whereas RP58 levels decreased after 24 hours of PDGF-BB stimulation compared to
FGF2-treated controls. In line with other BTB/POZ domain containing proteins, we found that
RP58 interacted directly with the HDAC-associated corepressor SMRT in biochemical assays. We
suggest that RP58 may play a role in the maturation of postmitotic neurons in dorsal forebrain
development.




                                                50            Developmental & Perinatal Biology 2006
                                                                                                  P14
SUBCELLULAR LOCALIZATION OF α-, β- AND γ-SECRETASES IN PRIMARY CELL
CULTURES OF HUMAN NEURONS AND GLIA CELLS

Johanna Sundqvist, Linda Csöregh, Eva Andersson and Gabriel Fried

Division of Obstetrics and Gynecology, Department of Women and Child Health, Karolinska
University Hospital Solna, Stockholm, Sweden


Alzheimer´s disease (AD) is one of the most common types of dementia, where senile plaques are a
general hallmark. These plaques are generated by deposition of the -amyloid peptide (A), which
is generated by proteolytic processing of the amyloid precursor protein (APP) by the enzymes -, -
and -secretase. The -secretase pathway yields neuroprotective soluble APP, while cleavage by -
secretase leads to formation of the A peptide. An alteration in APP processing, yielding more A
peptide, is regarded to be a central event in AD pathology.

We have examined the subcellular localization of TACE and ADAM10 (-secretases), BACE (-
secretase) and Presenilin-1 (a part of the -secretase complex) in primary cell cultures of human
fetal neurons and glia cells by immunofluorescence and confocal microscopy.

We have found immunoreactivity for TACE, ADAM10 and BACE in cells positive for both GFAP
(astrocyte marker) and III-tubulin (immature neuronal marker). TACE had pronounced perinuclear
localization in both neurons and glia cells, partly colocalized with GFAP and -tubulin. ADAM10
immunoreactivity was mostly seen in the nucleus in both neurons and glia cells, but also in some
perinuclear areas. BACE showed diffuse staining of the whole cell in glia cells, while neurons had
somewhat stronger staining in the nucleus together with diffuse staining in the rest of the cell body.
Presenilin-1 was localized in the cell bodies of both neuron and glia cells, with stronger staining in
the nucleus.

These results show that the APP-processing secretases TACE, ADAM10, BACE and Presenilin-1
are expressed in fetal neurons and glia cells from primary cell cultures. The localization patterns
were different, with TACE showing the most pronounced perinuclear staining pattern.




                                                    51            Developmental & Perinatal Biology 2006
                                                                                                    P15
THE ROLE OF ANGIOTENSIN II RECEPTORS AT THE MATERNAL-FETAL
INTERFACE AND THEIR INTERACTION WITH TRANSFORMING GROWTH
FACTOR β

Clare Tower, John Aplin and Philip Baker

Maternal and Fetal Health Research Centre, University of Manchester, Manchester, UK


Pre-eclampsia and fetal growth restrictionare devastating pregnancy complications, in which altered
trophoblast kinetics and invasion play a vital role. Although the mechanisms have yet to be defined,
cellular interactions at the maternal-fetal interface are paramount. Angiotensin II (AII), acting via its
receptors (type 1, AT1 and type 2, AT2) and transforming growth factor-β (TGFβ) are implicated in
both trophoblast invasion and the pathogenesis of these conditions. Interactions between these
factors have been described in other tissues, and we have evidence of a novel interaction between
TGFβ and the AT1 receptor in trophoblast.

TGFβs exert an inhibitory effect on trophoblast invasion. We hypothesize these effects may, at least
in part, be the result of actions on the renin-angiotensin system. AII is generated in the placenta and
inhibits invasion of a trophoblast cell line via the AT1 receptor. We have demonstrated that TGFβ1
increases AT1 receptor mRNA levels in a trophoblast cell line, which may enable greater AII
signalling. Furthermore, the AT1 gene promoter contains multiple binding sites for TGFβ signaling
molecules. AII may therefore represent a downstream mechanism by which TGFβ limits trophoblast
invasion. AII also acts via the AT2 receptor but data on the placental expression of these receptors
is contradictory. Therefore, we hypothesize that TGFβ may inhibit trophoblast invasion via
upregulation of the AII signalling pathway.
In order to investigate these hypotheses, the inhibitory action of AII on trophoblast invasion has
been confirmed on a further trophoblast cell line, SGHPL-4. Ongoing studies are examining the
action of AII on primary cytotrophoblasts and trophoblast outgrowth from first trimester placental
explants. To further investigate the action of TGFβ1 on AT1 receptor expression,
immunohistochemistry and Western Blotting assays are in progress. Definitive molecular studies
are investigating the presence of the AT2 receptor in placental villi and decidua. Subsequent
experiments will examine the co-localization of TGFβs and the AII receptors at the utero-placental
interface, interactions between them and actions on proliferation and apoptosis. These studies will
greatly advance our understanding of placental development. Given the impact of the intrauterine
environment on long-term health and disease, and the roles that TGFβ and the renin- angiotensin
systems play in cardiovascular and renal pathology, these findings may also be of interest to the
wider research community.




                                                     52             Developmental & Perinatal Biology 2006
                                                                                                  P16
ACTIVATION OF TEMPORAL CORTEX IN 8-MONTH OLD INFANTS AFTER VISUAL
EXPOSURE TO THEIR MOTHER’S FACE AND TO AN UNKNOWN FACE. A NEAR
INFRARED SPECTROSCOPY STUDY.

Jakob Carlsson, Marco Bartocci, Matteo Bruschettini and Hugo Lagercrantz

Neonatal Research Unit, Astrid Lindgren‟s Children‟s Hospital, Karolinska Institutet, Stockholm,
Sweden


Background and Aims
One characteristic that makes human brain so unique is its ability at recognizing. Thus far, little is
known about neural mechanisms underling the capacity to discrimine objects/faces in human
infants, mainly because there are few non-invasive techniques available.
In this study, we aimed to investigate, using a non-invasive technique such as near-infrared
spectroscopy (NIRS), cortical activation-related haemodynamic changes in response to visual
stimulation in 8-month old infants after exposure to a known and an unknown face.

Methods
Twelve infants have been monitored, two children have been excluded due to technical problems.
The mean age of the children was 8 months.
We have used 3 different types of visual stimuli: a grey background (GB), the photo of the mother‟s
face (MF) and the photo of an unknown person (UF). These images have been presented to the
child, on a PC screen, in the following order: GB – MF – GB – UF, keeping the environment
around the child as quite as possible. Changes in regional oxy [HbO2], deoxy-[HbH] and total
hemoglobin [Hb tot] have been measured by NIRS in both temporal and occipital areas on the right
side.

Results
In the temporal area, [HbO2] increased following the presentation of both MF, from 0 to 1,2 µmol/l
(p<0.001) and UF, from 0 to 1,0 µmol/l (p<0.001), comparing to GB. The increase in [HbO2] is
larger, when the child is presented to MF than it is when the child is presented to UF, from 1,2 to
1,0 µmol/l (p<0.05)
In the occipital area, there is no difference in [HbO2] following presentation of MF. There is a
decrease in [HbO2] following the presentation of UF (p<0.001)
During MF presentation, the children reacted with clear increase of their attention to the screen.

Conclusions
Cortical activation in response to the exposure to human faces was detected in 8-months old
children.
Right temporal cortex shows a typical activation pattern suggesting signal processing.
Right occipital cortex reacts by showing a typical deactivation pattern.
Temporal cortex appears to be involved in face recognition processes at this age.
A non invasive technique such as NIRS can be used to assess and follow the emergence and the
development of image processing in infants.


                                                    53            Developmental & Perinatal Biology 2006
Trainee Contacts:

Canada
Desai               Ridham     ridham@gmail.com
DiGiovanni          Valeria    valeria.digiovanni@utoronto.ca
Emack               Jeff       jeff.emack@utoronto.ca
Fornaro             Enrica     enrica.fornaro@inwind.it
Galtseva            Alevtina   alya.galtseva@utoronto.ca
Gill                Paul       paul.gill@utoronto.ca
Jain                Venu       vjain@mtsinai.on.ca
Jayasuriya          Kusala     kusala.jayasuriya@utoronto.ca
Johnson             Ben-Hur    benhurjohnsonm@hotmail.com
Jones               Michael    michael.jones@utoronto.ca
Juneja              Subhash    sjuneja@sickkids.ca
Kapoor              Amita      a.kapoor@utoronto.ca
Lai                 Ingrid     ilai@mshri.on.ca
Paltoo              Aarti      aarti.paltoo@utoronto.ca
Petropoulos         Sophie     sophie.petropoulos@gmail.com
Rac                 Valerija   valerija@mshri.on.ca
Thavandiran         Nimalan    nimalant@gmail.com

Sweden
Bergman             Gunnar     gunnar.bergman@akademiska.se
Carlsson            Jakob      jakob.carlsson.980@student.ki.se
Crambert            Susanne    susanne.crambert@ki.se
Ehnman              Monika     monika.ehnman@licr.ki.se
Johansson           Sofia      sofia.johansson@cmm.ki.se
Nisancioglu         Maya       maya.nisancioglu@ki.se
Olson               Linus      linus.olson@ki.se
Rohdin              Malin      malin.rohdin@ki.se
Sandberg            Magnus     magnus.sandberg@licr.ki.se
Sjöberg             Agneta     agneta.sjoberg@ki.se
Södersten           Erik       erik.sodersten@ki.se
Sundqvist           Johanna    johanna.sundqvist@ki.se
Wallenborg          Karolina   karolina.wallenborg@ki.se
Wallgard            Elisabet   elisabet.wallgard@ki.se

United Kingdom
Desforges           Michelle   m.desforges@postgrad.manchester.ac.uk
Hollis              Lisa       ljh502@soton.ac.uk
Mills               Tracey     tracey.mills@manchester.ac.uk
Sellayah            Dyan       dy180484@soton.ac.uk
Tower               Clare      clare.tower@manchester.ac.uk


                                    54            Developmental & Perinatal Biology 2006
Faculty Contacts:

Canadian

Dr. Lee Adamson         adamson@mshri.on.ca
Dr. John Challis        j.challis@utoronto.ca
Dr. Leigh Coultas       lcoultas@sickkids.ca
Dr. Caroline Dunk       dunk@mshri.on.ca
Dr. Robert Jankov       robert.jankov@sw.ca
Dr. John Kingdom        jkingdom@mtsinai.on.ca
Dr. Steve Lye           lye@mshri.on.ca
Dr. Steve Matthews      stephen.matthews@utoronto.ca
Dr. Michael Meaney      michael.meaney@mcgill.ca
Dr. Mira Puri           mira.puri@swri.ca
Dr. Sue Quaggin         quaggin@mshri.on.ca
Dr. Janet Rossant       janet.rossant@sickkids.ca
Dr. Ian Scott           ian.scott@sickkids.ca
Dr. Cheryle Seguin      cseguin@sickkids.ca
Dr. Marla Sokolowski    msokolow@utm.utoronto.ca
Dr. Neil Sweezey        neil.sweezey@utoronto.ca
Dr. Keith Tanswell      keith.tanswell@sickkids.ca


Swedish
Dr. Ola Hermanson       ola.hermanson@cmb.ki.se
Dr. Hugo Lagercrantz    hugo.lagercrantz@ki.se
Dr. Thomas Ringstedt    thomas.ringstedt@ki.se
Dr. Karl Tryggvason     karl.tryggvason@ki.se


United Kingdom
Dr. Ian Crocker         ian.crocker@manchester.ac.uk
Dr. Rebecca Lee Jones   rebecca.lee.jones@manchester.ac.uk




                                    55            Developmental & Perinatal Biology 2006
                Developmental and Perinatal Biology 2006

                                        Course Evaluation
Please complete, remove and return the evaluation. This is important because it will help us in the
design of future courses. Thank you.

Trainee from: (please tick) Karolinska Institute _____             University of Toronto _____
                            United Kingdom _____

Lecture Course:          (please circle one)

1)     Pregnancy and Birth:
Overall       could be improved                1    2      3       4       5       excellent

       a) Dr. R. Lee Jones                     1    2      3       4       5
       b) Dr. C. Dunk                          1    2      3       4       5
       c) Dr. I. Crocker                       1    2      3       4       5
       d) Dr. J. Kingdom                       1    2      3       4       5


2)     Pulmonary & Renal Development:
Overall       could be improved                1    2      3       4       5       excellent

       a) Dr. K. Tryggvason                    1    2      3       4       5
       b) Dr. K. Tanswell                      1    2      3       4       5
       c) Dr. R. Jankov                        1    2      3       4       5



3)     Cardiovascular Development and Function:
Overall       could be improved                1    2      3       4       5       excellent

       a) Dr. M. Pira                          1    2      3       4       5
       b) Dr. L. Coultas                       1    2      3       4       5
       c) Dr. I. Scott                         1    2      3       4       5
       d) Dr. L. Adamson                       1    2      3       4       5



                                                   56            Developmental & Perinatal Biology 2006
4)     Neurodevelopment:
Overall       could be improved            1      2      3          4    5       excellent

       a) Dr. H. Lagercrantz               1      2      3          4    5
       b) Dr. O. Hermanson                 1      2      3          4    5
       c) Dr. T. Ringstedt                 1      2      3          4    5
       d) Dr. S. Matthews                  1      2      3          4    5


5)     Embryo Patterning and Organogenesis:
Overall       could be improved            1      2      3          4    5       excellent

       a) Dr. J. Rossant                   1      2      3          4    5
       b) Dr. C. Sequin                    1      2      3          4    5
       c) Dr. S. Quaggin                   1      2      3          4    5


6)     Developmental Origins of Health and Disease:
Overall       could be improved            1      2      3          4    5       excellent

       a) Dr. M. Sokolowski                1      2      3          4    5
       b) Dr. J. Challis                   1      2      3          4    5
       c) Dr. M. Meaney                    1      2      3          4    5




Please outline how you feel the lecture course could be improved:




                                                 57            Developmental & Perinatal Biology 2006
Practical Courses:
Which practical courses did you attend (1 indicates 1st course, 2 indicates 2nd course)
      1.      Endocrine Gene Expression                      _____
      2.      Genetic Manipulation of Embryos                _____
      3.      New Techniques in Mouse Physiology             _____
      4.      Lung Physiology/Cell Biology                   _____
      5.      Imaging Technologies                           _____


1st Course
Course Content:
             could be improved                1      2       3       4       5       excellent

Course Organization:
                                              1      2       3       4       5



2nd Course
Course Content:
             could be improved                1      2       3       4       5       excellent

Course Organization:
                                              1      2       3       4       5



How could the practical courses be improved:




Social Program:
               could be improved              1      2       3       4       5       excellent

Could the social program/accommodations be improved:




                                                    58             Developmental & Perinatal Biology 2006
Overall Course:
             could be improved           1      2      3     4       5       excellent

How do you think the course could be improved next year:




                                    Thank You




                                               59          Developmental & Perinatal Biology 2006

								
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