Spring Meeting of the BSCDB, jointly organized with FWO-...(PCP) genes celsr2/off-road and frizzled3a/off-limits block FBMN caudal migration. Here, we investigated the role of cadherins

BSCDB & FWO & FEDRA – March 2009 – Leuven “CELL FATE DETERMINATION IN THE EMBRYO”

Spring Meeting of the BSCDB, jointly organized with FWO-

Flanders (Network on Cell-Cell and Cell-Matrix Interactions)

and FEDRA (IUAP/PAI6 Network 20)

"Cell Fate Determination in the Embryo"

30 – 31 March 2009

Provinciehuis, Leuven, B

Local organizers

Guido David and Danny Huylebroeck


The meeting is sponsored by


BSCDB Spring meeting 2009 Leuven POSTER PRIZES At the end of the Spring Meeting of 2009, four poster prizes were awarded to

• Lydie Flasse (U.Liège) • Anne-Christine Hick (UCL Brussels) • Tim Pieters (UGent – VIB) • Stijn Van Dyck (KULeuven)

For their posters: DECIPHERING THE MOLECULAR CASCADE GOVERNING PANCREATIC DEVELOPMENT IN ZEBRAFISH : ROLE OF Neurod AND Rfx6 Lydie Flasse, Isabelle Manfroid, Gérard Gradwohl, Bernard Peers and Marianne Voz Lab. Molecular Biology and Genie Genetique, GIGA, Ulg, 4000 Liège STROMAL CELL-DERIVED FACTOR-1 CONTROLS BRANCHING MORPHOGENESIS IN THE PANCREAS AND SUBMANDIBULAR GLANDS Anne-Christine Hick (1), Jonathan M. van Eyll (1), Sabine Cordi (1), Lara Passante (2), Takashi Nagaswa (3), Pierre Vanderhaeghen (2), Pierre J. Courtoy (1), Guy G. Rousseau (1), Frédéric P. Lemaigre (1) and Christophe E. Pierreux (1) 1: Université catholique de Louvain and de Duve Institute, Brussels, Belgium 2: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Free University of Brussels, Brussels, Belgium 3: Institute for Frontier Medical Science, Kyoto, Japan A TRANSGENIC APPROACH TO DISSECTING THE FUNCTIONS OF THE P120CTN ISOFORMS IN VIVO Tim Pieters (1,2), Petra D'hooge (1,2), Anja Lambrechts (3,4), Frans van Roy (1,2) and Jolanda van Hengel (1,2) 1: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium 2: Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium 3: Department of Medical Protein Research, VIB, B-9052 Ghent, Belgium 4: Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium THE ROLE OF THE SYNTENIN/SYNDECAN INTERACTION IN ZEBRAFISH EPIBOLY Stijn Van Dyck, Kathleen Lambaerts, Elke Vermeiren and Pascale Zimmermann Laboratory for Signal Integration in Cell Fate Decision, Department of Human Genetics, K.U.Leuven, B-3000 Leuven, Belgium


Monday, March 30, 2009

From 8:15 Registration

9:15-9:30 Welcome (Danny Huylebroeck)

All sessions take place in the “Auditorium” – poster session, coffee breaks, lunches in ”Spoor 95”, Coffee Corner and Cobbaert Corner

SESSION 1: EARLY DEVELOPMENTAL DECISIONS

Chair: Susana Chuva de Sousa Lopes and Danny Huylebroeck

9:30 - 10:15

Jean-Paul Vincent (National Institute for Medical Research, London, UK)

Control of positional information and growth by Wingless

10:15 - 11:00

Christof Niehrs (German Cancer Research Center, Heidelberg, Germany)

Regulation of Wnt signaling in vertebrate development

11:00 – 11:30

Coffee break

SESSION 2: DEVELOPMENT OF THE NERVOUS SYSTEM (EARLY SPECIFICATION)

Chair: Eric Bellefroid and Bassem Hassan

11:30 - 12:15

Nancy Papalopulu (University of Manchester, UK)

Cell polarity and fate in primary neurogenesis

12:15 - 13:00

Magdalena Götz (LMU, München, and Helmholtz Zentrum, Neuherberg, Germany)

Neurogenesis from glial cells: molecular and cellular mechanisms

13:00 – 14:30

Lunch and poster session

SESSION 3: DEVELOPMENT OF THE NERVOUS SYSTEM (LATE SPECIFICATION)

Chair: André Goffinet and Pierre Vanderhaeghen

14:30 - 15:15

Selected oral presentation 1

Joke Debruyn, Eve Seuntjens and Danny Huylebroeck

SIP1 is a regulator of wnt signaling during early mid/hindbrain patterning


Yibo Qu, Libing Zhou, Aurélia Ravni, André M. Goffinet and Fadel Tissir

CELSR1, 2, and 3 genes differentially regulate direction and trajectory of facial branchiomotor neuron

migration in the mouse


15:15 - 16:00

François Guillemot (National Institute for Medical Research, London, UK)

Transcriptional control of neurogenesis and neuronal migration

16:00 - 16:45

Alain Chédotal (Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, UMR, Paris,

France)

Rewiring hindbrain commissures

16:45

Reception

Tuesday, March 31, 2009

All sessions take place in the “Auditorium” – poster session, coffee breaks, lunches in ”Spoor 95”, Coffee Corner and Cobbaert Corner

SESSION 4: DEVELOPMENT OF THE VASCULAR SYSTEM AND HEMATOPOIESIS

Chair: Mieke Dewerchin and An Zwijsen

09:15 - 10:00

Elaine Dzierzak (Erasmus University Medical Center, Rotterdam, The Netherlands)

Positional information in hematopoietic stem cell development

10:00 - 10:45

Joerg Wilting (Centre of Anatomy, Department of Anatomy and Cell Biology, Göttingen, Germany)

Lymph hearts and heart lymphatics

10:45 -11:15

Coffee break

11:15 - 12:00

Stefan Schulte-Merker (Hubrecht Institute, Utrecht, The Netherlands)

Genetic analysis of zebrafish lymphangiogenesis

12:00 - 12:45


Lieve Umans*, Iván Moya*, Ward Sents, Elke Maas, Danny Huylebroeck and An Zwijsen

Inactivation of SMAD1 and SMAD5 in endothelial cells demonstrates that the BMP SMADS are essential in

angiogenesis and lymphangiogenesis


Peggy Raynaud, Christophe E. Pierreux, Aline Antoniou, Marco Pontoglio and Frédéric P. Lemaigre

Bile duct development proceeds according to a new mode of tubulogenesis, a link to ciliogenesis

12:45 - 14:00

Lunch and poster session


SESSION 5: DEVELOPMENT OF THE ENDODERMAL DERIVATIVES

Chair: Frederic Lemaigre and Bernard Peers

14:00 - 14:45

Anne Grapin-Botton (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland)

Generating cell diversity in the mouse pancreas

14:45 - 15:30

Silvia Cereghini (CNRS - Université Pierre et Marie Curie, Paris, France)

Gene regulatory networks involved in liver and pancreas specification in mice

15:30 - 16:00

Coffee Break

16:00 - 16:45

Didier Stainier (Cardiovascular Research Institute, UCSF, San Francisco, USA)

Endodermal organ development and regeneration in zebrafish

16:45 - 17:15

Award of the BSCDB poster prizes

Oral presentation 1 Debruyn Joke

presenting author ; e-mail : [email protected]

SIP1 IS A REGULATOR OF WNT SIGNALLING DURING EARLY MID/HINDBRAIN PATTERNING

Joke Debruyn (1), Eve Seuntjens (1,2) and Danny Huylebroeck (1)

1: Lab. Molecular Biology, Campus Gasthuisberg O&N, K.U.Leuven, B-3000 Leuven 2: Institute of Neurosciences, Developmental Neurobiology Unit, U.C.Louvain The adult central nervous system (CNS) is an incredibly complex structure that controls basically all the workings of the body. During development, the entire CNS arises from a single cell layered neuroepithelium, called the neural plate. This neural plate is established by neural induction around embryonic day (E)7.5 in mouse development. Already early during development several organizing centres are formed within the neural plate and these organizers instruct surrounding cells to either differentiate or proliferate. One of these organizing centres is the mid-hindbrain organizer (MHB), which is located within the neural plate in between future mid- and hindbrain. The MHB is characterised by expression of Wnt1 on its anterior and Fgf8 on its posterior side and this organizer is essential for the patterning of both future mid –and hindbrain. Sip1 (Smad Interacting Protein-1) is a transcriptional repressor that is expressed throughout the developing neural plate. Sip1 knockout embryos show morphological defects in the neural plate starting from embryonic day (E)8.5 and die around E9.5. Both morphological analysis and whole mount in situ hybridisation experiments show that a neural plate is formed and initial forebrain, midbrain and hindbrain territories are present in Sip1 knockout embryos. However, we found a dramatic increase in Dkk1 expression in the future midbrain of these embryos already at E8.0. Dkk1 is a potent inhibitor of the Wnt signalling cascade and since Wnts are known to play important patterning roles during early mid-hindbrain development, we analysed in detail the expression domains of known midbrain or hindbrain markers. In line with disturbed Wnt signalling, we observed that several known mid-hindbrain markers like En2 and Pax5 are not expressed in their correct domains in the future mid-hindbrain of Sip1 knockout embryos. Sip1 thus seems to be essential for early mid-hindbrain patterning. Since Sip1 generally acts as a transcriptional repressor, we suspected that Dkk1 might be a direct target of Sip1. Luciferase reporter assay data indeed indicate that the Dkk1 gene is a direct target of Sip1. We can thus conclude that Sip1 plays an important role early mid-hindbrain patterning, most probably by regulation of Dkk1 gene expression and thereby indirectly controlling Wnt signalling. ( = Poster abstract 15 )

BSCDB & FWO & FEDRA – March 2009 - Leuven Debruyn Joke "CELL FATE DETERMINATION IN THE EMBRYO"

Oral presentation 2 Qu Yibo


CELSR1, 2, AND 3 GENES DIFFERENTIALLY REGULATE DIRECTION AND TRAJECTORY OF FACIAL BRANCHIOMOTOR NEURON MIGRATION IN MOUSE

Yibo Qu, Libing Zhou, Aurélia Ravni, André M. Goffinet, Fadel Tissir

Université catholique de Louvain, 1200 Brussels During hindbrain development, facial branchiomotor neurons (FBMN) migrate from paramedial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity (PCP) genes celsr2/off-road and frizzled3a/off-limits block FBMN caudal migration. Here, we investigated the role of cadherins Celsr1, 2, and 3, as well as Fzd3 in FBMN migration in mice. In Celsr1 mutants, FBMN caudal migration was compromised and neurons often migrated rostrally from r4 into r2 and r3, a migration defect never observed previously in any vertebrate. This phenotype was not seen in Celsr1|Islet1 mice, indicating that the effects of Celsr1 on FBMN migration are non cell autonomous. In Celsr2 mutants, FBMN migrated abnormally and prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3, and mimicked by mutation of Fzd3. In contrast to Celsr1, Celsr3 acted cell autonomously, because anomalies of FBMN migration were identical in Celsr2 -/-,Celsr3 -/- embryos, and in Celsr2-/-,Celsr3|Islet1 embryos. In double Celsr1 -/-,Celsr2-/- embryos, the FBMN migration phenotype was similar to that in Celsr2 -/-, indicating that Celsr2 is epistatic to Celsr1. These data suggest that Celsr1-3 regulate FBMN migration is two different ways. On one hand, Celsr1 specifies the direction of FBMN migration along the rostrocaudal axis, whereas on the other hand Celsr2, Celsr3 and Fzd3 regulate the trajectory of the migrating neurons. ( = Poster abstract 38 )

BSCDB & FWO & FEDRA – March 2009 - Leuven Qu Yibo "CELL FATE DETERMINATION IN THE EMBRYO"

Oral presentation 3 Umans Lieve and Moya Ivan

presenting author ; e-mail : [email protected], [email protected]

INACTIVATION OF SMAD1 AND SMAD5 IN ENDOTHELIAL CELLS DEMONSTRATES THAT THE BMP SMADS ARE ESSENTIAL IN ANGIOGENESIS AND LYMPHANGIOGENESIS

Lieve Umans (*), Ivan Moya (*), Ward Sents, Elke Maas, Danny Huylebroeck and An Zwijsen

Laboratory of Molecular Biology (Celgen), Division Molecular and Developmental Genetics, Department of Human Genetics (DME) and

Department of Molecular and Developmental Genetics (VIB11), Faculty of Medicine, University of Leuven

*: These authors contributed equally to this work Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor beta (Tgf-beta) superfamily. Smad5 and Smad1 are highly homologous and they mediate primarily bone morphogenetic protein (Bmp) signals. The early embryonic lethality of Smad5 and Smad1 deficient mice obstructs further investigation at later stages of development. Endothelial specific deletion of only Smad1 (personal communication) or Smad5 (Umans et al., 2007) results in viable mice without any morphological defect. The comparable expression patterns of both genes and the phenotype of Smad1+/-Smad5+/- embryos suggest that both genes can functionally compensate for each other. Therefore we have generated Tie-2-Cre Sm1fl/fl Sm5f/fl mice to study the role of both BMP-Smads in blood vessel development. Endothelial specific Smad1/Smad5 embryos die between E9.5 and E10.5 of embryonic development due to cardiovascular defects. Embryos with only one functional Smad1 or Smad5 allele, however, can survive until E12.5-E14.5 which enables us to investigate the role of Smad1 and Smad5 in angiogenesis at later stages of embryonic development. In E8.5 Tie-2-Cre Sm1fl/fl, Sm5fl/fl embryos the vascular plexus is formed in the yolk sac but one day later the yolk sacs have highly dilated blood vessels and show clear defects in vascular remodeling and branching. The embryo proper has an enlarged amniotic cavity, an enlarged pericardial cavity, only a rudimentary heart tube is formed and in the whole embryo a clear defect in the capillary network is visible. Histological analysis of those embryos demonstrates that the heart is underdeveloped, has a reduced amount of trabeculae, the endocardial layer is detached from the myocardial layer and in some embryos there is invasion of the endocardial layer into the compact myocardial layer. The main vessels (dorsal aorta, cardinal vein) are formed demonstrating that both in the yolk sac and in the embryo vasculogenesis is not impaired by Smad1/Smad5 deficiency. At E9.5 a clear defect in the capillary network in the head and the dorsal anastomoses is observed in the KO embryos demonstrating that there is a primary role for Smad1/Smad5 in vessel patterning and sprouting (angiogenesis). Additional experiments will be performed to analyze more in detail the role of Smad1/Smad5 in this process. A detailed marker analysis in embryos with one functional Smad1 or Smad5 allele demonstrated that at later stages partial deletion of Smad1/5 in endothelial cells not only causes cardiovascular defects but also defects in lymphangiogenesis. At E12.5-E14.5 the embryos are oedematic and histological analysis demonstrates that the lymphatic sacs are enormously enlarged. This phenotype will be analyzed more in detail in the next year. Together these data demonstrate that the BMP Smads are essential both in angiogenesis and lymphangiogenesis. ( = Poster abstract 44 )

BSCDB & FWO & FEDRA – March 2009 - Leuven Umans Lieve and Moya Ivan "CELL FATE DETERMINATION IN THE EMBRYO"

Oral presentation 4 Raynaud Peggy


BILE DUCT DEVELOPMENT PROCEEDS ACCORDING TO A NEW MODE OF TUBULOGENESIS, A LINK TO CILIOGENESIS

Peggy Raynaud (1), Christophe E. Pierreux (1), Aline Antoniou (1), Marco Pontoglio (2) and Frédéric P. Lemaigre (1)

1: de Duve Institute and Université catholique de Louvain, Brussels, Belgium 2: Pasteur Institute/CNRS URA 2578, Paris, France Dysfunctions of primary cilia are implicated in a class of human inherited diseases commonly referred to as ciliopathies. Despite their clinical and pathological heterogeneity, these disorders share altered regulations of tubular morphogenesis which mainly affect the kidneys and the liver. To understand the mechanisms of dysmorphogenesis in diseased livers, we analyzed the development of bile ducts in normal mouse embryos, and in two mouse models (Hnf6-/- and Hnf1beta-/- mice) with biliary dysgenesis, and focused on the acquisition of polarity and on expression of ciliopathic genes in the biliary epithelial cells. In wild-type livers, the bile ducts develop from the ductal plate. The latter is composed of a single layer of biliary precursor cells which forms in between the portal mesenchyme and the liver parenchyma. We found that at this stage, the ductal plate cells are partly polarized as indicated by the localization of basolateral markers. Then, at specific areas lumen open between the biliary precursors and the hepatoblasts. Therefore, at that stage, the developing ducts constitute asymmetrical tubular structures, which are lined on the parenchymal side by hepatoblasts and on the portal side by biliary precursors of the ductal plate. Concomitantly to lumen formation tight junctions become apparent. Progressively, cells on the parenchymal side of the ductal epithelium fully polarize, giving rise to a mature ducts. Based on these observations, we propose that bile duct development occurs via a new mode of tubulogenesis characterized by the transition through an asymmetrical state. We have shown earlier that, in the pancreas, HNF6 controls a network of genes involved in ciliogenesis (1), and that the expression of HNF1-beta is controlled by HNF6 in the liver (2,3). We looked at biliary cell polarization and cilia formation in HNF6- and HNF1-beta-deficient livers. In both mouse models cilia were absent and polarity was perturbed, as indicated by the mislocalization of the Golgi apparatus, the basal body and the basal lamina. We also measured the expression of ciliopathic genes, and found that the expression of Cys1 is strongly diminished in HNF6- and HNF1-beta-deficient livers. We conclude that bile ducts develop according to a new mode of tubulogenesis characterized by transient asymmetry. This morphogenic process implies stepwise aquisition of polarity and ciliogenesis, and is controlled by HNF6 and HNF1-beta. 1: Pierreux CE et al. Gastroenterology 130, 532-541, 2006 2: Clotman F. et al. Development 129, 1819-1828, 2002 3: Coffinier C. et al. Development 129, 1829-1838, 2002 ( = Poster abstract 40 )

BSCDB & FWO & FEDRA – March 2009 - Leuven Raynaud Peggy "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 1 Aceto Jessica


THE FUNCTION OF SOX4 TRANSCRIPTION FACTORS IN ZEBRAFISH BONE DEVELOPMENT AND HOMEOSTASIS

J. Aceto (1), P. Motte (2), J.A. Martial (1) and M. Muller (1)

1: Molecular Biology and Genetic Engineering, University of Liège, B-4000 Sart-Tilman 2: Plant Cellular Biology, University of Liège, B-4000 Sart-Tilman The Sox4 transcription factor belongs to the group C of the SRY-like HMG-box family and is involved in mammalian development of the endocardial crest, brain, lungs, teeth, gonads, pancreas and lymphocytes. Recently, heterozygous KO mice for Sox4 were shown to present a decrease in bone mass and mineralization. Moreover, the presence of Sox4 is required for expression of specific markers in osteoblast cultures. Thus, we decided to investigate the role of Sox4 in bone development. In zebrafish, two homologs for the mammalian Sox4 are present, sox4a and sox4b. sox4b was shown to be required for glucagon cell development in zebrafish pancreas. Here, we investigate the role of the sox4a and sox4b genes in cartilage and bone development in zebrafish. Therefore, we focus on the first bone structures to be formed, the cranial cartilage and bones. We show by in situ hybridization that both genes are expressed in the pharyngeal region, albeit at different periods during development. Furthermore, sox4a and sox4b knock-down leads to morphological modifications in the jaw. Our study suggests that both genes appear to be important for the correct formation of the mandible and hyoid. Further experiments will determine the exact pharyngeal tissues expressing the zebrafish sox4 genes as well as their exact function. In addition, in order to discover new factors involved in bone formation, we investigate the changes in gene expression induced by bone-remodelling chemicals or microgravity after one day treatment by microarray analysis. Major differences are observed between different treatments leading to similar outcomes in bone formation after 5 days.

BSCDB & FWO & FEDRA – March 2009 - Leuven Aceto Jessica "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 2 Altomare Claudia


CARDIAC-TYPE CALCIUM RELEASE MECHANISM IN CARDIAC PRECURSOR CELLS

C. Altomare (1), L. Barile (2), S. Marangoni (1), M. Rocchetti (1), G. Mostacciuolo (1), A. Giacomello (2), E. Messina (2) and A. Zaza (1)

1: Biotechnologies and Biosciences Department , University of Milano Bicocca, p.za della Scienza 2, 20126 Milano, Italy

2: Department of Experimentel Medicine University of Rome "La Sapienza", viale Regina Elena, 324 00161 Roma, Italy

Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). CSs provide a suitable environment for coordination of cell–cell contact, self-organization, differentiation, and electrical properties, all of which are essential for the identity of cells capable of heart repair. While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca2+ release is a shared property of many cell types, Ca+ release through RyR channels is muscle specific. Aims: To test if cardiac-type Ca2+ release mechanism is present in CPCs and whether it may develop during the CSs stage. Methods: Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca2+ transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca2+ release from intracellular stores was tested by brief exposures to caffeine (CAF, 10 mM) or ATP (200 μ,M) respectively in Ca2+-free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis. Results: In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %, p

Poster 3 Andries Vanessa


A CONSTITUTIONAL TRANSLOCATION T(1,17)(p36.2,q11.2) IN A NEUROBLASTOMA PATIENT DISRUPTS NBPF1, A NOVEL PUTATIVE TUMOR SUPPRESSOR GENE

Vanessa Andries (1,2), Karl Vandepoele (1,2), Katrien Staes (1,2) and Frans van Roy (1,2)

1: Department for Molecular Biomedical Research, VIB 2: Department of Biomedical Molecular Biology, Ghent University Technologiepark 927, B-9052 Ghent, Belgium Neuroblastoma (NB) is the most common extracranial solid tumor in children and is characterized by a number of recurrent genetic alterations: gain of chromosome 17q, amplification of MYCN, and deletion of 1p36. We found that a constitutional translocation t(1,17)(p36.2,q11.2) in a neuroblastoma patient resulted in the disruption of a novel gene, NBPF1 (Neuroblastoma Breakpoint Family, member 1). This gene is built of repetitive elements and is subject of structural variation in the human population. Thorough analysis of genomic sequences revealed that NBPF1 is a member of a recently expanded gene family, with gene copies located on segmental duplications of chromosome 1. Both in silico and in vitro analysis failed to identify any rodent orthologs for the human NBPF genes. The members of the NBPF gene family are widely expressed, both in normal and cancerous tissues, including neuroblastoma cells. Expression profiling of NBPF1 in a panel of neuroblastoma cell lines showed that expression of NBPF1 is significantly lower in cell lines with 1p deletion than in cell lines with a normal 1p chromosome, making NBPF1 a viable candidate as a tumor suppressor gene in neuroblastoma. Transfection experiments revealed a cytoplasmic localization of the different NBPF proteins. Constitutive overexpression of different NBPF paralogs resulted in cell death in a variety of cell lines, including MCF7/AZ, HEK293T, and the neuroblastoma cell line IMR-32. We use now a conditional expression system to circumvent the detrimental properties of the overexpressed NBPF proteins and to investigate the process leading to NBPF1-induced cell death. In a preliminary in vivo experiment, expression of NBPF1 decreased proliferation of xenograft tumors. Additional experiments will be performed to validate this important finding and to establish the molecular basis of the observed results.

BSCDB & FWO & FEDRA – March 2009 - Leuven Andries Vanessa "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 4 Bammens Leen


CHARACTERIZATION OF THE PEN-2 KNOCK OUT MOUSE

Leen Bammens, An Herreman, Véronique Hendrickx and Bart De Strooper

Laboratory for Neuronal Cell Biology and Gene Transfer, Departement of Molecular and Developmental Genetics, VIB11, KU Leuven and Flanders Interuniversitary Institute for Biotechnology , Leuven , Belgium

Gamma-secretase is a key player in the pathogenesis of Alzheimer’s disease. This aspartic protease cleaves many type I membrane proteins, including the amyloid precursor protein (APP). The cleavage of APP results in the formation of Abeta peptides, the main constituent of the amyloid plaques that characterize Alzheimer’s disease. Gamma-secretase is composed of 4 subunits which are all required for activity: Presenilin which contains the catalytic site and Nicastrin, Aph-1 and PEN-2 whose functions remain largely unknown. We generated a PEN-2 knock out (KO) mouse model which provides us with an excellent tool to study the function of PEN-2. To answer the question whether PEN-2 has other functions besides a role in gamma-secretase activity, we will characterize PEN-2 KO embryos and compare them to Presenilin double KO embryos, which completely lack gamma-secretase activity. The PEN-2 knock out genotype is lethal, so we will also determine the point of embryonic lethality. Furthermore, we will characterize the morphology of the embryos by EM microscopy to verify whether somites are still present in PEN-2 KO embryos. Moreover, using in situ hybridization techniques, we will verify the processing of Notch and in the fibroblasts derived from the PEN-2 KO embryos, we will investigate the APP processing by looking at the production of Abeta species.

BSCDB & FWO & FEDRA – March 2009 - Leuven Bammens Leen "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 5 Bardet Sylvia M.


A MOUSE/CHICK REVISED MODEL OF DORSOVENTRAL ORGANIZATION OF THE SECONDARY PROSENCEPHALON IN THE DEVELOPING EMBRYO BASED ON MOLECULAR MARKER PATTERNS

Sylvia M. Bardet (2), Jose Luis E. Ferrán (1), Luisa Sánchez-Arrones (1), Juan E. Sandoval (1), Margaret Martínez-de-la-Torre (1), John L.R. Rubenstein (3) and Luis Puelles (1)

1: Human Anatomy department, faculty of medicine, Campus of Espinardo, Murcia, Spain

2: Molecular and Neurobiology Cellular Center, University of Liège, Sart Tilman, Liege, Belgium

3: Genetics, Development and Behavioral Sciences Building, University of California at San Francisco, USA

Brain development during embryogenesis implies the progressive generation of morphogenetic fields by gene network, and leads to a same general histogenetic schema named Bauplan shared by all vertebrates. The prosomeric model (Puelles and Rubenstein 1993, 2004) divides the forebrain into the same transverse segments (prosomeres) and longitudinal zones (floor, basal, alar and roof plates) in all vertebrates, based on morphological and gene pattern observations. The secondary prosencephalon, located rostral to the prethalamus (prosomere 3), includes the hypothalamus, the eye field and the telencephalic vesicles and is considered as the rostral extremity of the brain axis. The particularity of the hypothalamus resides in its special developmental position closed to the precordal plate, which is able to affect through numerous factors the development of many structures, as the adenohypophysis, the neurohypophysis, the eye fields, the mammillary region... In this manner, the development of the hypothalamus remains relatively unclear because of its complexity. In this study, we explored -through RNA in situ hybridization and immunohistochemistry methods- the embryonic expression of molecular markers (Dlx, Nkx, Six, Shh, Otp, DBX, SF1, Pax, …) in chick and mouse embryos. We propose a new relevant model of the organization of the secondary prosencephalon, more consistent, which better explains the longitudinal subdomains of the secondary prosencephalon, considered as a protosegment with a special basal plate. The hypothalamic basal plate looks like a “half-opened fan”, with the caudal part dorsoventrally thinner and the rostral part more expanded, which led in the past to misunderstanding of these “oblique” domains, in particular the mammillar region. These findings form a starting point to better understand the development of the hypothalamus and the telencephalon, which is to this day misinterpreted concerning the neuraxis and the brain orientation. Supported by Grant from University of Liège to S.B.

BSCDB & FWO & FEDRA – March 2009 - Leuven Bardet Sylvia M. "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 6 Beukelaers Pierre


A SPECIFIC REQUIREMENT FOR CDK6 DURING POSTNATAL HIPPOCAMPAL NEUROGENESIS

Pierre Beukelaers (1,*), Renaud Vandenbosch (1,*), Nicolas Caron (1), Laurence Borgs (1), Laurent Nguyen (1), Shibeshih Belachew (1,2), Gustave Moonen (1,2), Hiroaki Kiyokawa (3), Mariano. Barbacid (4), David Santamaria (4) and Brigitte Malgrange (1)

1: GIGA-Neurosciences, University of Liege, C.H.U. Sart Tilman, 4000 Liège, Belgium 2: Department of Neurology, University of Liege, C.H.U. Sart Tilman, 4000 Liège,

Belgium 3: Department of Molecular Pharmacology and Biological Chemistry, Robert H. Lurie

Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA 4: Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas,

Madrid, Spain *: contributed equally In mammals, the dentate gyrus (DG) of the hippocampus, with the subventricular zone (SVZ) of the lateral ventricles, is the only region where neurogenesis persists throughout entire life. At the present time, the intrinsic mechanisms driving cell cycle progression in the DG are poorly understood. During cell cycle progression, advancement through G1 phase is tightly controlled by two members the cyclin-dependent kinase (Cdk) family, Cdk4 and its close cousin Cdk6, which only differ by their tissue specificity and/or their differential activation during development. Here, we investigate the specific role of these two proteins during postnatal dentate gyrus neurogenesis. Using immunohistochemistry directed against Cdk4 or Cdk6, we first demonstrated that Cdk4 was not expressed in the DG at P30. Conversely, at the same stage, we observed that Cdk6 was specifically expressed in the subgranular zone (SGZ), the germinative zone of the DG. Furthermore, virtually all Ki67-positive proliferating cells of the SGZ express Cdk6. We then compared the number of proliferating cells in the SGZ at P30 between WT and Cdk4-/- or Cdk6-/- mice. Whereas we did not detect any difference between WT and Cdk4-/- mice, we showed that deletion of Cdk6 results in a more than 50% reduction the number of proliferating cells in the SGZ. These data suggest that postnatal hippocampal neurogenesis, at the level of cell proliferation, is differentially controlled by Cdks. Indeed, whereas Cdk4 appears not to play any role, Cdk6 seems to be crucial in this specific context. This study is one of the first which clearly shows that Cdk4 and Cdk6 are not redundant in all tissues.

BSCDB & FWO & FEDRA – March 2009 - Leuven Beukelaers Pierre "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 7 Binot Anne-Catherine


NKX6 GENES CONTROL ENDOCRINE PANCREATIC CELL FATE IN ZEBRAFISH

Anne-Catherine Binot (1), Isabelle Manfroid (1), Marie Winandy (1), Patrick Motte (2), Bernard Peers (1) and Marianne Voz (2)

1: Unit of Molecular Biology and Genetic Engineering, University of Liège, GIGA-R, B34, Avenue de l’Hôptial 1, 4000 Liège, Belgium

2: Laboratory of Plant Cell and Molecular Biology, Department of Life sciences, Institut of Botany, University of Liège, 4000 Liège, Belgium

The homeodomain proteins Nkx6.1 and Nkx6.2 have been shown to be expressed in the murine pancreas and deficiency for Nkx6.1 results in a specific abrogation of beta-cell neogenesis while the pancreas develops normally in Nkx6.2 single-mutant mice. In contrast, Nkx6.1/Nkx6.2 double-mutant embryos display a severe reduction in alpha-cell number. In zebrafish, nkx6.1 and nkx6.2 are expressed at the beginning of the somitogenesis in the endoderm before to be restricted to the pancreas. First expressed in the same cells, their expression domains rapidly segregate, nkx6.1 being expressed more ventrally. While nkx6.1 is never expressed in the mature hormone-producing endocrine cells, nkx6.2 is expressed in beta-cells until at least 24hpf. The knock-down of nkx6.1 or of nkx6.2 leads to a striking decrease of the number of glucagon-expressing cells, while the expression of insulin, somatostatin or ghrelin is not affected. In contrast, the double knock-down of the two genes results in a clear decrease of insulin expression as well as in a almost complete disappearance of glucagon expression. One hypothesis we are currently investigating is that the global dose of nkx6 proteins present in the embryo is critical for pancreatic cell fate: a high dose of nkx6 would be necessary to allow alpha-cell differentiation while lower amount would be required to permit beta-cells to differentiate.

BSCDB & FWO & FEDRA – March 2009 - Leuven Binot Anne-Catherine "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 8 Bruyère Françoise


NEW INSIGHT INTO THE IMPLICATION OF PROTEASES IN LYMPHANGIOGENESIS BY USING A NEW 3D CULTURE SYSTEM

Françoise Bruyère, Laurence Melen-Lamalle, Silvia Blacher, Benoît Detry, Jean-Michel Foidart and Agnès Noël

Laboratory of Tumor and Development Biology, Center for Experimental Cancer Research (CECR), Center for Biomedical Integrative Genoproteomics (CBIG), University of Liège, B-4000 Liège, Belgium

Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few data’s are available concerning their role in lymphangiogenesis. Previously, we successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiment of lymphedema. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

BSCDB & FWO & FEDRA – March 2009 - Leuven Bruyère Françoise "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 9 Cassano Marco


MAGIC FACTOR-1: A NEW ENGINEERED PROTEIN INVOLVED IN MUSCULAR HYPERTROPHY AND REGENERATION

Marco Cassano (1), Mattia Quattrocelli (1), Ilaria Perini (1), Flavio Ronzoni (2), Matilde Bongio (1), Silvio Conte (2), Daniela Galli (2), Gabriella Cusella De Angelis (1) and Maurilio Sampaolesi (1, 2)

1: Translational Cardiomyology, SCIL KULeuven Herestraat 49, B-3000 Leuven, Belgium

2: Human Anatomy, University of Pavia, Via Forlanini 8, I-27100 Pavia, Italy Background: Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues. Adult myogenic precursor cells (satellite cells) express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells. Methodology/Results: Magic-F1 (Met-Activating Genetically Improved Chimeric Factor-1) is an HGF-derived, engineered protein that contains two Met-binding domains. Hemizygous Magic-F1 transgenic mice displayed constitutive muscular hypertrophy, improved running performance and accelerated muscle regeneration following injury. We followed the transgene expression during embryogenesis (E8.5 to E17.5) by in situ hybridization and we found that Magic-F1 is expressed in bone cartilage primordium, somite dorsal side, tail and limb bud. These data confirm that Magic F1 is expressed in embryo muscle precursors, suggesting a role of this factor in muscle development possibly triggering the downregulation of Pax3 signal pathway in skeletal muscle precursor cells. Moreover, Magic-F1 ameliorates the dystrophic phenotype of alpha-sarcoglycan null mice, a model of muscular dystrophy, as measured by both anatomical/histological analysis and functional tests of mice subjected to adenovirus mediated Magic-F1 gene delivery. We analysed also omozygous Magic-F1 mice by morphometric and physiological tests that demonstrate a higher size of the muscle fibers and a better running performance. Preliminary data suggest a possible implication of the engineered protein in development of the vascular network, increasing the capillary vessel number. Conclusions: Because of these features Magic-F1 represents a novel molecular tool to counteract muscle wasting in major muscular diseases such as cachexia or muscular dystrophy.

BSCDB & FWO & FEDRA – March 2009 - Leuven Cassano Marco "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 10 Cludts Stéphanie


MACROPHAGE MIGRATION INHIBITORY FACTOR CORRELATES WITH RECURRENCE AND SURVIVAL IN PATIENTS WITH HEAD AND NECK CARCINOMA

Stéphanie Cludts (1), Dominique Chevalier (2), Sabine André (3), Xavier Leroy (4), Hans-Joachim Gabius (3), Christine Decaestecker (5) and Sven Saussez (1,6)

1: Lab. of Anatomy & Cell. Biology, FMP, UMH, Mons, Belgium 2: Dept. of ORL, Hôp. C. Hurriez, Lille, France 3: Inst. of Physiol. Chem., Fac. of Vet. Med., L-M-University, Munich, Germany 4: Hôp. C. Hurriez & Centre de Biologie-Pathologie-CHRU, Lille, France 5: Lab. of Toxicol., Inst. of Pharm., ULB, Brussels, Belgium 6: Dept. of ORL, ULB, Brussels, Belgium Objective: Macrophage migration inhibitory factor (MIF), originally identified as a product of activated lymphocytes, has since been found to play an important role in the host response to endotoxic shock, joint inflammation, glomerulonephritis, transplantation rejection, and atherosclerosis. In addition to its potent effects on the immune system, several reports have also linked MIF to fundamental processes that control cell proliferation, differentiation, angiogenesis, tumour progression and tumour immune escape. Related to these later involvements, the aims of the present study are 1) to examine MIF expression in relation to neoplastic progression of head-and-neck squamous cell carcinoma (HNSCC) {by comparing normal epithelia (N_E) to low-grade dysplasia (Low_D), high-grade dysplasia (High_D) and carcinomas (CA)}, 2) to investigate whether a relationship exists between MIF expression and tumor differentiation, clinicopathologic features as well as long-term prognosis. Materials and Methods: Using computer-assisted microscopy, the immunohistochemical expression of MIF was quantitatively evaluated on a series of 62 laryngeal SCCs. Using Western blotting, MIF expression was also determined on a series of 20 HNSCC fresh biopsies. ELISA was done to assess the MIF serum level in a series of 77 HNSCC patients and 20 healthy volunteers. Results: Our data revealed an association between MIF expression levels and neoplastic progression of HSCCs. The MIF levels were significantly higher in CA than in N_E, Low_D or High_D. Finally, high levels of MIF expression were associated with rapid recurrence rates and dismal prognoses in our series of laryngeal SCCs. HNSCC patients displayed higherlevels of circulating MIF. Conclusions: For the first time (at least to the best of our knowledge) these results demonstrate that LSCCs presenting high levels of MIF expression have worse prognoses than those with low MIF levels. In addition, the quantitative determination of MIF expression in LSCCs acts as an independent prognostic marker when opposed to TNM staging.

BSCDB & FWO & FEDRA – March 2009 - Leuven Cludts Stéphanie "CELL FATE DETERMINATION IN THE EMBRYO"



GALECTIN-8 CORRELATES WITH TUMOR PROGRESSION IN PATIENTS WITH HEAD AND NECK CARCINOMA

Stéphanie Cludts (1), Dominique Chevalier (2), Sabine André (3), Xavier Leroy (4), Hans-Joachim Gabius (3), Christine Decaestecker (5) and Sven Saussez (1,6)

1: Lab. of Anatomy & Cell. Biology, FMP, UMH, Mons, Belgium 2: Dept. of ORL, Hôp. C. Hurriez, Lille, France 3: Inst. of Physiol. Chem., Fac. of Vet. Med., L-M-University, Munich, Germany 4: Hôp. C. Hurriez & Centre de Biologie-Pathologie-CHRU, Lille, France 5: Lab. of Toxicol., Inst. of Pharm., ULB, Brussels, Belgium 6: Dept. of ORL, ULB, Brussels, Belgium Introduction: Galectin-8 belongs to the family of tandem-repeat type galectins. It consists as several isoforms, each made of two domains of ~ 140 amino-acids, both having a carbohydrate recognition domain (CRD). Galectin-8, like other galectins, is a secreted protein. Upon secretion galectin-8 acts as a physiological modulator of cell adhesion. The levels of expression of this galectin positively correlate with certains human cancers. The galectin-8 overexpression might give to these cancers some growth ans metastasis related advantages due to its ability to modulate cell adhesion and cellular growth. Related to these later involvements, the aims of the present study are to examine galectin-8 expression in relation to neoplastic progression of head-and-neck squamous cell carcinoma (HNSCC) {by comparing normal epithelia (N_E) to low-grade dysplasia (Low_D), high-grade dysplasia (High_D) and carcinomas (CA)} Materials and Methods: Using computer-assisted microscopy, the immunohistochemical expression of galectins-8 was quantitatively evaluated on a series of 74 hypopharyngeal SCCs and 44 laryngeal SCCs. Results: Our data revealed an association between galectins-8 expression levels and neoplastic progression of laryngeal and hypopharyngeal SCCs. The galectin-8 levels were significantly higher in CA than in N_E, Low_D or High_D in both types of HNSCCs. The staining profile is mostly significant in the cytoplasm rather than in the nucleus. Conclusions: These results demonstrate the presence of a relation between galectin-8 levels and the neoplastic progression of LSCCs and HSCCs.




GALECTIN FINGERPRINTING IN WARTHIN’S TUMORS: LECTIN-BASED APPROACH TO TRACE ITS ORIGIN?

Stéphanie Cludts (1), Laurence Deleval (2), Christine Decaestecker (3), Nicolas Sirtaine (4), Anaelle Duray (1), Perle Ernoux (1), Sabine André (6), Hans-Joachim Gabius (6), Myriam Remmelinck (7), Xavier Leroy (5) and Sven Saussez (1)

1: Lab. of Anat. & Cell. Biol., FMP, UMH, B-Mons 2: Dpt. of Pathol., ULg, B-Liège 3: Lab. of Toxicol., Inst. of Pharm.,ULB, B-Brussels 4: Dpt. of Pathol., Inst. Bordet, ULB, B-Brussels 5: Dept. of Pathol., Fac. of Med., CHRU, F-Lille 6: Inst. of Physiol. Chem., Fac. of Vet. Med., L-M-U, D-Munich 7: Dpt. of Pathol., ULB, Hôp. Erasme, B-Brussels Warthin’s tumor of the parotid gland is assumed to originate from proliferation of epithelial inclusions to parotid lymph nodes. In that case, these cells are supposed to retain characteristics similar to common salivary gland ductal cells. Using an immunohistochemical fingerprinting with four members of the family of adhesion/growth-regulatory galectins and comparison to intra- and interlobular ducts, marked similarities were noted for presence of galectins-3, -7 and -8. Notably, profiles of lectin binding, determined by applying human lectins as probes, were also similar when testing biotinylated galectins-3 and -8. Besides defining the galectin histochemical parameters in Warthin’s tumors this study adds support to the hypothesis of heterotopia.


Poster 13 Crabbe Ellen


GASTRULATION MOVEMENTS IN THE XENOPUS EMBRYO ARE DEPENDENT ON THE INTERACTION OF THE CELL-CELL ADHESION MOLECULE N-CADHERIN WITH THE WASP-FAMILY MEMBER WAVE-1

Ellen Crabbe (1,2), Mieke Delvaeye (1,2), Griet Van Imschoot (1,2), Malgorzata Ciesiolka (1,2), Frans Van Roy (1,2) and Kris Vleminckx (1,2)

1: Department for Molecular Biomedical Research, VIB 2: Department of Biomedical Molecular Biology, Ghent University Technologiepark 927, B-9052 Ghent, Belgium Actin polymerization and reorganization are key events in several processes such as cell motility, cell polarity, cytokinesis, cell adhesion, etc. The small GTPases Rho, Rac and Cdc42 are important modulators of the actin cytoskeleton and act upstream of various effector proteins. One group of RhoGTPase effectors is the family of the Wiskott - Aldrich syndrome Proteins (WASPs) which contains 5 members: WASP, N-WASP, and three WAVE proteins (WAVE-1, -2, -3). The WASP proteins regulate actin dynamics by binding to and activating the Arp2/3 complex. WAVE proteins act downstream of Rac1 and have been found to form a heterocomplex with four other proteins, which are essential for activation of the ARP2/3 complex. We have identified WAVE-1 as a novel interaction partner of the cell-cell adhesion molecule N-cadherin. WAVE proteins (especially WAVE-1 and WAVE-2) have already been described to be indispensable for the maintenance and stabilization of E-cadherin-mediated cell-cell adhesion in epithelial cells. Loss of WAVEs resulted in fewer lamellipodia and improperly formed adhesive bonds. Strickingly, we found that N-cadherin -in strong contrast to E-cadherin- had a negative effect on the level of WAVE1 in 293T cells. This downregulation was also observed for the other members of the WAVE complex. Conversely, depletion of N-cadherin by siRNA resulted in an upregulation of WAVE-1 levels. Moreover, depletion of N-cadherin in Xenopus laevis embryos resulted in an increase of XWAVE-1 levels and caused extreme gastrulation defects. These gastrulation defects could be rescued by co-depletion of XWAVE-1, indicating that the gastrulation defects induced by N-cadherin depletion are indeed the result of elevated levels of XWAVE-1. Since N-cadherin is known to have a pro-migratory effect on certain cells, we postulate that this is at least partially mediated by affecting the levels of WAVE proteins, possibly leading to a loss of stable cell-cell contacts and hence promoting invasion.

BSCDB & FWO & FEDRA – March 2009 - Leuven Crabbe Ellen "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 14 Dalcq Julia


FUNCTION OF THE EGR1 TRANSCRIPTION FACTOR IN ZEBRAFISH CARTILAGE DEVELOPMENT

Julia Dalcq, Vincent Pasque, Marc Muller and Joseph A. Martial

Laboratory of Molecular Biology and Genetic Engineering , GIGA Research, B34 1st floor, B-4000 Liège

The cartilaginous elements that form the pharyngeal arches of the zebrafish derive from cranial neural crest cells. The proper patterning and morphogenesis of neural crest-derived cartilages are regulated by reciprocal interactions between neural crest cells and other tissue types such as pharyngeal endoderm, ectoderm and mesoderm. We study the zebrafish transcription factor Egr1 (Early growth response 1) involved, among others, in pituitary development, wound healing and cancer. We determined the expression pattern of egr1 by in situ hybridization in wild-type and mutant embryos, our results indicate that egr1 is expressed among others in the pharyngeal endoderm. Our functional studies (knock down and rescue experiments) reveal that Egr1 is absolutely required for cranial cartilage morphogenesis and differentiation. To examine more in detail the mechanisms by which Egr1 acts on the formation of cranial cartilages, we analyzed the expression of different marker genes involved in pharyngeal development in egr1 morphants using in situ hybridization. Egr1 does not act on neural crest cell formation and migration, nor on endoderm formation. In contrast, the absence of Egr1 leads to abolition of expression of sox9b in endoderm and runx2b in cartilage. It has been previously described that the transcription factor Sox9b acts on runx2b expression by an extracellular signalling pathway. We are presently investigating various candidate molecules that could be involved in signalling between pharyngeal endoderm and primordial cartilage.

BSCDB & FWO & FEDRA – March 2009 - Leuven Dalcq Julia "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 16 Delval Stephanie


ON THE MODE OF ACTION OF THE HOXA1 TRANSCRIPTION FACTOR IN MAMMARY CARCINOGENESIS IN THE MOUSE

Stéphanie Delval (1), Cécile Lallemand (2) and René Rezsohazy (1)

1: Unit of Veterinary Sciences, UCL, B-1348 Louvain-la-Neuve 2: Laboratory of Tumor and Development Biology, ULg, B-4000 Liège Hoxa1 belongs to the homeotic or Hox gene family that encodes transcription factors involved in patterning embryonic territories and governing organogenetic processes. Recently, Hoxa1 has been reported to display oncogenic activity and to be overexpressed in the mammary gland in response to a deregulation of the autocrine growth hormone (GH). It has further been suggested that Hoxa1 plays a pivotal role in mammary carcinogenesis under those conditions of GH misregulation. While the functions of Hoxa1 have been well characterized, its mode of action under normal and pathologic situations remains poorly documented. In that regard, the aim of this work is to investigate the way Hoxa1 is active in mammary carcinomas. The Hoxa1 protein possesses a hexapeptide motif required for its interaction with Pbx transcription factors. Replacement of the WM amino acids by AA in the hexapeptide (Hoxa1 WM-AA mutant) results in a loss of Hoxa1 function during embryonic development. This supports that the normal embryonic activity of Hoxa1 relies on its partnership with Pbx. We now address the importance of this partnership for the oncogenic activity of Hoxa1. Expression vectors for Hoxa1 WM-AA and wild type Hoxa1 have been stably transfected in human breast MCF7 cells. These cells are transformed but keep several epithelioid characters so that they define a suitable model to study key processes of oncogenesis in vitro. Cellular clones have been obtained and are now under characterization for proliferation, resistance to apoptosis, anchorage-independent growth and contact inhibition,… While the Hoxa1 wild type protein induces enhancement of cell proliferation upon constitutive expression, this is not the case for Hoxa1 WM-AA. Our data are therefore indicative that interaction between Hoxa1 and Pbx might be critical for mammary carcinogenesis. “Knock-in” mice harbouring the WM-AA substitution in Hoxa1 are currently treated with DMBA to induce mammary carcinogenesis involving GH signalling. We will address to what extent the Hox-Pbx interaction is required for Hoxa1-mediated carcinogenesis in vivo. We will therefore check if induced tumors in wild type mice actually express Hoxa1 and further assess if mutant mice show decreased carcinoma incidence or less aggressive carcinogenesis.

BSCDB & FWO & FEDRA – March 2009 - Leuven Delval Stephanie "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 17 Detry Benoît


ROLE OF MATRIX METALLOPROTEASE-12 IN CHOROIDAL NEOVASCULARIZATION AND CORNEAL LYMPHANGIOGENESIS

Benoît Detry (1), Vincent Lambert (1, 2), Julie Lecomte (1), Françoise Bruyère (1), Laurence Melen-Lamalle (1), Vincent Dive (3) and Agnès Noël (1)

1: Laboratory of Tumor and Development Biology, GIGA-Cancer, ULg, Belgium 2: Service of Ophthalmology, CHU, Liège, Belgium 3: CEA, Commissariat à l’Energie Atomique, Service d’Ingénierie Moléculaire de

Protéines (SIMOPRO), CE-Saclay, 91191 Gif/Yvette, Cedex, France Matrix metalloproteases (MMPs) constitute a family of endopeptidases implicated in physiological and pathological processes such as development, wound healing, angiogenesis and tumour growth. MMP-12 is mainly produced by macrophages and is associated with chronic obstructive pulmonary diseases, and other inflammatory pathologies. It is potentially anti-angiogenic by cleaving plasminogen leading to the formation of angiostatin, a strong angiogenesis inhibitor. We are studying the role of this protease in a murine model of laser-induced choroidal neovascularisation (CNV), in which angiogenesis and inflammation are particularly implicated, and in which different other proteases play an important role. To assess the specificity of MMP-12 effect on choroidal angiogenesis, we extended our investigation to corneal lymphangiogenesis, which is associated to corneal inflammation and risk of graft rejection after cornea transplantation. We show by semi-quantitative RT-PCR analysis and immunohistochemistry that MMP-12 is upregulated from 5 days after CNV induction. Compare to their wild type controls, mice deficient for MMP-12 display a reduced neovascular reaction evidenced on flat mounted choroids and a reduced lesion size on tissue sections 14 days after treatment. The deposition of basement membrane and smooth muscle cells/pericytes around the neovessels is similar in MMP-12 KO mice and in control mice. Immunohistochemistry analysis of inflammatory cells shows a reduced number of CD11b positive cells and neutrophils inside the lesions of MMP-12 KO mice as compared to controls. The use of a specific MMP-12 inhibitor, subcutaneously delivered, reduces the neovascular area and the lesion size as compared to the control group, and so, restores the phenotype of MMP-12 deficient mice. In a model of corneal lymphangiogenesis induced by thermal cauterization, we show no difference between MMP-12 KO and WT mice, and the specific inhibition of MMP-12 in this model don’t modify the lymphangiogenic response, suggesting no role of MMP-12 in this process. Our data suggest that MMP-12 could contribute to the development of laser-induced CNV by a pro-inflammatory role and that MMP-12 could be an interesting target for pharmacological treatment. However, MMP-12 doesn’t seem to be implicated in corneal lymphangiogenesis. These results are important for clinical investigation and underlie the need to develop different targets to inhibit angiogenesis in ocular diseases.

BSCDB & FWO & FEDRA – March 2009 - Leuven Detry Benoît "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 18 Djiotsa Joachim


MIR-375 IN ZEBRAFISH PANCREAS DEVELOPMENT

Djiotsa J., Martial J.A., Peers B., Voz M.L., Manfroid I.

GIGA-Research - Unité de Biologie Moléculaire et Génie Génétique, 1 avenue de l'Hôpital, Tour GIGA, B34, Université de Liège, B-4000 Sart Tilman, Belgium

MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that regulate gene expression. A recent study showed that knockdown of 11 miRNAs conserved between zebrafish and mammals do not result in visible defects except for miR-375 (Kloosterman et al., 2007). miR-375 displays a conserved specific expression in the pancreas and pituitary and its knockdown in zebrafish embryos results in aberrant migration of pancreatic endocrine cells. In order to better understand the role of miR-375, we analyzed in detail its expression in the zebrafish embryonic pancreas and its function by gain- and loss-of-function approaches. By double fluorescent in situ hybridization, we showed that miR-375 is expressed in the endocrine pancreas and more precisely in the hormone expressing cells while being excluded from the progenitors. . miR-375 knock-down of zebrafish embryos reveal that the pancreatic endocrine cells normally aggregate in a single islet by 24 hpf but then they start to disperse from 35hpf onwards in the morphants. In addition, a progressive decrease of the insulin protein level is observed. Overexpression of miR-375 RNA duplex in embryos leads to a duplication of the pancreas and, generally, of endodermal organs normally presenting a left-right asymmetry. The heart, derived from the mesoderm, also exhibits left-right asymmetry defects. The implication of miR-375 in the molecular processes governing left-right asymmetry is currently investigated. Our attention is first concentrated on the Nodal and BMP pathways since the list of the zebrafish miR-375 targets predicted by Miranda comprised several genes of these pathways, providing putative candidates to test. Their implication in formation of the endocrine pancreas will be assessed. In parallel, other potential candidate miR-375 target genes will be identified by a bioinformatic search focusing on conserved miR-375 targets. References: Wigard P Kloosterman, Anne K Lagendijk, René F Ketting, Jon D Moulton, Ronald H A Plasterk (2007). Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development. PLoS Biol. Jul 24,5 (8)

BSCDB & FWO & FEDRA – March 2009 - Leuven Djiotsa Joachim "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 19 Dobreva Mariya P.


AMNION PLASTICITY: LESSONS FROM THE MOUSE

Mariya P. Dobreva (1), Paulo N.G. Pereira (1), Danny Huylebroeck (2) and An Zwijsen (1)

1: Laboratory of Developmental Signaling 2. Laboratory of Molecular Biology (Celgen) Department of Molecular and Developmental Genetics, VIB and Center for Human

Genetics, K.U. Leuven, Leuven, Belgium To survive and develop in utero, a mammalian conceptus will develop beside the embryonic body per se a large portion of extraembryonic supporting tissues and organs. These appendages are shed at birth. The amnion is the innermost extraembryonic membrane that surrounds the foetus of most amniotes and delineates the fluid filled amniotic cavity. Next to its function in in utero development, the amnion has already for several decades been shown to have important clinical applications, such as its use as a dressing to stimulate and improve epithelial wound healing in skin and ocular wounds. The last few years, several research groups have reported that cell populations from human term amnion have pluripotent differentiation ability and stem cell features. Amnion development and the putative origin of the amniotic stem cells has been described surprisingly poorly and conflicting interpretations exist. Furthermore, the existence of such cells with stem cell properties in mouse amnion is sill unknown. Here, we highlight the similarities and dissimilarities in the early development of the human and mouse amnion, and discuss data from genetic mouse models that may shed light on the origin and the maintenance of amniotic stem cells. Smad5 is an intracellular mediator of BMP signaling. We have shown that Smad5 mediated signaling is essential for amnion homeostasis in the mouse. Its deficiency results in a local thickening of the amnion and formation of cell aggregates, which is followed by ectopic vasculogenesis and haematopoiesis, and ultimately even ectopic appearance of primordial germ cell-like/stem cell-like cells in the aggregates in a regionalized fashion. As the appearance of these lineages is known to be BMP dependent, it was hypothesized that a gradient of BMP signaling, but also interplay between BMP, Wnt and Nodal signaling pathways, may determine the fate of these cells. To check this hypothesis we have developed an amnion explant system. Mid-gestation wild type mouse amnion in culture shows responsiveness to BMP signaling after BMP4-stimulation, as shown by upregulation of the expression of BMP-target genes on mRNA level. We will discuss the further evaluation of this explant system. Wild type freshly isolated mouse amnion from mid-gestation also expresses mRNA for BMPs, BMP-receptors and BMP-target genes. As in mouse is possible to study amnion development from the moment of its formation untill term, and different genetic models can be used, these insights from the mouse may be useful for understanding the underlying molecular mechanisms of amnion plasticity.

BSCDB & FWO & FEDRA – March 2009 - Leuven Dobreva Mariya P. "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 20 Ernoux-Neufcoeur Perle


DETECTION OF HUMAN PAPILLOMAVIRUS AND RELEVANT TUMOR SUPPRESSORS IN HYPOPHARYNGEAL SQUAMOUS CELL CARCINOMAS

Perle Ernoux-Neufcoeur (1), Mohammad Arafa (2), Christine Decaestecker (3), Xavier Leroy (4), Philippe Delvenne (2) and Sven Saussez (1,5)

1: Lab. of Anat. & Cell. Biol., FMP, UMH, B-Mons 2: Dpt. of Pathol., ULg, B-Brussels 3: Lab. of Toxicol, Inst. of Pharm., ULB, B-Brussels 4: Hosp. Claude Huriez & center of Biol.-Pathol. – CHRU, F-Lille 5: Dpt. of Oto-Rhino-Laryngol., Université Libre de Bruxelles, ULB, B-Brussels Background : Human papillomavirus (HPV) DNA has been detected in approximately 15% of all head and neck carcinomas. Recent studies have shown a predilection of HPV for oropharyngeal squamous cell carcinomas (OSCC). Moreover, a subgroup of OSCC patients characterized by HPV DNA-positive and p16-expressing tumors presented better prognosis (lower recurrence rate and improved disease free survival). We sought to determine the prevalence of HPV in a homogeneous series of 75 stage IV hypopharyngeal SCCs. The E6 and E7 genes of the oncogenic HPVs encode oncoproteins that bind and degrade p53 and retinoblastoma (Rb) tumor suppressors, respectively. Rb dowregulation resulted in p16 upregulation. Methods: Using polymerase chain reaction (PCR) amplification with GP5+/GP6+ primers consensus and specific primers for HPV 6, 11, 16, 18, 31, 33, 39, 42, 44, 51, 52, 56, 59, we studied the HPV status in a series of 75 stage IV HSCCs with long-term patient follow-up. The level of cellular proteins (p16 and p53) was examined by immunohistochemistry. Results: 72% of our HSCC samples contained HPV DNA determined by the primer consensus GP5+/GP6+. Among this HPV+ subgroup, we identified 12 patients with high-risk HPV (type 16, 18, 51, 52, 56, 59) and 6 patients with low-risk HPV (type 42, 44). The HPV+ and high-risk HPV+ subgroup presented lower rate of metastatic nodes (75% of HPV+ patients were N+ versus 100% of HPV- patients) and lower rate of capsular effraction (44% of HPV+ patients presented nodes with capsular effraction versus 85% of HPV- patients). The 5-year disease free survival was 73% in p53- tumors versus 48% in p53+ tumors (p=0.008). The 5-year disease free survival was 100% in p16+ tumors (only 5 cases) versus 58% in p16- tumors. Conclusion: Using the combination of the HPV status and the cellular expression of p16 and p53 in a series of stage IV HSCCs, we identified a subgroup of patients HPV+/p16+/p53- presenting a better prognosis.

BSCDB & FWO & FEDRA – March 2009 - Leuven Ernoux-Neufcoeur Perle "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 21 Espana Agnès


ROLES OF THE ONECUT TRANSCRIPTION FACTORS IN CATECHOLAMINERGIC NEURON DIFFERENTIATION

Agnès Espana and Frédéric Clotman

Neural differentiation, Hormone and Metabolic Research Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium

The Onecut (OC) transcription factors, namely HNF-6, OC-2 and OC-3, are characterized by a single cut domain and a divergent homeodomain. They have been shown to regulate morphogenesis and differentiation in liver and pancreas, as well as differentiation of spinal motor neurons. They have also been detected in the encephalon during murine embryonic development, but the identity of the cells wherein they are expressed and the roles they play in these cells remain unknown. Here, we identified by immunofluorescence neuronal populations wherein OC proteins are present and we studied the roles of the OC in these cells by analyzing fetuses single- or double-knockout for the OC factors. Our study revealed that the OC are expressed in populations of postmitotic differentiating neurons in the diencephalon, in the midbrain and in the hindbrain. We then focused on catecholaminergic neurons and we observed that, at embryonic day (e)14.5, OC factors are exclusively expressed in the A13 nucleus, a dopaminergic neuron population located in the zona incerta in the diencephalon. The study of e14.5 OC knockout fetuses showed that the expression of tyrosine hydroxylase (TH), an enzyme required for dopamine biosynthesis, was abolished in the A13 nucleus of Hnf6-/-Oc2-/- fetuses. Surprisingly, another catecholaminergic population was affected in the Hnf6-/-Oc2-/- fetuses. Indeed, TH expression was lost in the Locus Coeruleus (LC), a noradrenergic neuron population located in the pons, although no expression of OC factors was observed in these cells. Interestingly, OC expression was detected in an adjacent group of cells characterized by the expression of Isl1, and the expression of Isl1 was lost in most of these cells in the Hnf6-/-Oc2-/- fetuses, suggesting an non cell-autonomous role of the OC factors in LC development. These results show that the OC factors are essential regulators of the differentiation of catecholaminergic neuron populations in the encephalon.

BSCDB & FWO & FEDRA – March 2009 - Leuven Espana Agnès "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 22 Flasse Lydie


DECIPHERING THE MOLECULAR CASCADE GOVERNING PANCREATIC DEVELOPMENT IN ZEBRAFISH : ROLE OF Neurod AND Rfx6

Lydie Flasse, Isabelle Manfroid, Gérard Gradwohl, Bernard Peers and Marianne Voz

Lab. Molecular Biology and Genie Genetique, GIGA, Ulg, 4000 Liège One of the goals of our laboratory is to decipher the molecular cascade governing pancreatic endocrine development in zebrafish. Ngn3 being a key factor for the induction of the endocrine cell differentiation program, we have focused our attention on two targets of Ngn3: neuroD and rfx6. In zebrafish, the bHLH transcription factor neurod starts to be expressed in the pancreatic area after gastrulation and is still detectable at 3 days of development (dpf). Knock down of neurod expression through morpholino injection leads to a complete lack of glucagon and ghrelin expressing cells, a slight reduction in the number of somatostatin cells while the number of insulin cells is not changed. Expression of the transcription factors isl1, nkx6.1 and nkx6.2 is also perturbed in neuroD morphants. We are currently analysing other pancreatic markers in order to place neuroD in the pancreatic cascade. rfx6, a winged helix factor, is restricted to the pancreatic area in zebrafish. Its expression starts at 16s stage and still persist at 3 dpf. Like neuroD knock down, rfx6 morphants display a complete absence of glucagon and ghrelin expressing cells while the number of insulin cells is not changed. However, the phenotype of rfx6 morphants is more severe as a drastic reduction in the number of somatostatin expressing cells is observed together with the dispersion of the insulin cells. Preliminary expression analysis of the transcription factors suggests that rfx6 is essential for the differentiation of the pancreatic precursors into mature hormone producing cells.

BSCDB & FWO & FEDRA – March 2009 - Leuven Flasse Lydie "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 23 Francius Cedric


THE ONECUT TRANSCRIPTIONAL ACTIVATORS HNF-6, OC-2 AND OC-3 ARE REQUIRED FOR PROPER DIFFERENTIATION OF THE SPINAL MOTOR NEURONS

Cédric Francius and Frédéric Clotman

Neuro-differentiation Lab (NEDI), Institute of Neurosciences (INES), Université Catholique de Louvain (UCL), B-1200 Brussels

Spinal motor neurons (MN) arise from a discrete population of progenitors located in the ventral part of the spinal cord. Progenitors first acquire a generic MN phenotype. Then, they further differentiate into distinct MN populations, i.e. somatic MN that form the medial motor column (MMC) and innervate the muscles of the body wall, visceral MN which gather in the preganglionic column (PGC) and innervate visceral organs, and, at the limb levels, somatic MN that migrate to the lateral motor column (LMC) and innervate the muscles of the limbs. The Onecut (OC) transcriptional activators, so called HNF-6, OC-2 and OC-3, are transiently expressed during the first steps of MN differentiation. Here, we study the roles of the OC factors during MN development. Consequences of the loss-of-function of OC factors were evaluated in transgenic mice single- or double-knockout for the OC genes. Gain-of-function was analyzed after overexpression of the OC factors in chick embryonic spinal cord. Inactivation of the OC factors affected the differentiation of each spinal MN population. Indeed, in the absence of HNF-6, the amount of MMC neurons was reduced without any increase in cell death. In Hnf6/Oc2 double-knockout embryos, PGC neurons were present but the repertoire of differentiation markers they expressed was altered. In addition, the MN located in the medial LMC (LMCm), which normally innervate the ventral half of the limbs, did not express LMCm markers but instead expressed markers of the lateral LMC (LMCl), which normally innervate the dorsal half of the limb. Accordingly, innervation of the ventral half of the limbs was affected in Hnf6/Oc2 double-knockout fetuses. Gain-of-function experiments to identify the mechanisms controlled by the OC factors during MN differentiation are ongoing. Thus, the OC transcriptional activators exert partially redundant functions that are required for proper differentiation of each population of spinal MN.

BSCDB & FWO & FEDRA – March 2009 - Leuven Francius Cedric "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 24 Fuchs Uta


FUNCTIONAL ANALYSIS OF HUMAN DELTA-PROTOCADHERIN 11X/Y (PCDH11X/Y)

Uta Fuchs (1,2) and Frans van Roy (1,2)

1: Department for Molecular Biomedical Research, VIB 2: Department of Biomedical Molecular Biology, Ghent University Technologiepark 927, B-9052 Ghent, Belgium Cadherins form a large superfamily of calcium-dependent cell adhesion molecules present in vertebrates and invertebrates (Hulpiau and van Roy, 2009). They are essential for cell-cell interactions and play critical roles in diverse biological processes such as cell recognition, signalling and morphogenesis. One subfamily of the cadherins is comprised by the clustered and non-clustered protocadherin (Pcdh) genes. The recently described delta-protocadherins belong to the non-clustered group and are divided into delta1- and delta2-protocadherins, a distinction that is mainly dependent on the unique structure of their cytoplasmic domain (Redies et al., 2005). An interesting member of the delta1-protocadherins is the human PCDH11X/Y gene pair, which is located in the non-pseudoautosomal X-Y homologous region Xq21.3/Yp11.2 (Yoshida and Sugano, 1999). Surprisingly, PCDH11Y does not exist in other species besides man, even not in other mammals including non-human primates, which has fed the hypothesis of a hominid specific role for PCDH11 in brain development (Wilson et al., 2006). However, very little is known about the function and the intracellular distribution of this delta1-protocadherin. Here we describe the intracellular localization of different isoforms of PCDH11X/Y in vitro. Furthermore, we are presently investigating how PCDH11X/Y and associated proteins influence cellular processes implicated in morphogenesis and tissue architecture. To this end, putative interaction partners of PCDH11X/Y were identified by use of the Mammalian Protein-Protein Interaction Trap (MAPPIT). Most interactions seem to be isoform-specific and are the subject of our further investigations. Hulpiau, P. and van Roy, F. (2009) Int. J. Biochem. Cell Biol., 41, 343-369. Redies, C., Vanhalst, K. and van Roy, F. (2005) Cell. Mol. Life Sci., 62, 2840-2852. Wilson, N.D., Ross, L.J., Crow, T.J., and Volpi, E.V. (2006). Cytogen. Genome Res. 114, 137-139. Yoshida, K., and Sugano, S. (1999). Genomics 62, 540-543.

BSCDB & FWO & FEDRA – March 2009 - Leuven Fuchs Uta "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 25 Garcia Marie Isabelle


LGR5 DEFICIENCY DEREGULATES WNT SIGNALING AND LEADS TO PRECOCIOUS PANETH CELL DIFFERENTIATION IN THE FETAL INTESTINE

Marie Isabelle Garcia (1), Mariangela Ghiani (1), Anne Lefort (1), Frédérick Libert (1), Sandra Strollo (1) and Gilbert Vassart (1,2)

1: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles, 1070 Brussels, Belgium

2: Department of Medical Genetics, Erasme Hospital, Faculty of Medicine, Université Libre de Bruxelles, 1070 Brussels, Belgium

The orphan Leucine-rich repeat G protein-coupled receptor 5 (LGR5/GPR49), a target of Wnt signaling, is a marker of adult intestinal stem cells (SC). However, neither its function in the adults, nor during development of the intestine have been addressed yet. In this report, we investigated the role of LGR5 during ileal development by using LGR5 null/LacZ-NeoR knock-in mice. X-gal staining experiments showed that, after villus morphogenesis, Lgr5 expression becomes restricted to dividing cells clustered in the intervillus region and is more pronounced in the distal small intestine. At day E18.5, LGR5 deficiency leads to premature Paneth cell differentiation in the small intestine without detectable effects on differentiation of other cell lineages, nor on epithelial cell proliferation or migration. Quantitative RT-PCR experiments showed that expression from the LGR5 promoter was upregulated in LGR5-null mice, pointing to the existence of an autoregulatory negative feedback loop in intact animals. This deregulation was associated with overexpression of Wnt target genes in the intervillus epithelium. Transcriptional profiling of mutant mice ileums revealed that LGR5 function is associated with expression of SC and SC niche markers. Together, our data identify LGR5 as a negative regulator of the Wnt pathway in the developing intestine.

BSCDB & FWO & FEDRA – March 2009 - Leuven Garcia Marie Isabelle "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 26 Hick Anne-Christine


STROMAL CELL-DERIVED FACTOR-1 CONTROLS BRANCHING MORPHOGENESIS IN THE PANCREAS AND SUBMANDIBULAR GLANDS

Anne-Christine Hick (1), Jonathan M. van Eyll (1), Sabine Cordi (1), Lara Passante (2), Takashi Nagaswa (3), Pierre Vanderhaeghen (2), Pierre J. Courtoy (1), Guy G. Rousseau (1), Frédéric P. Lemaigre (1) and Christophe E. Pierreux (1)

1: Université catholique de Louvain and de Duve Institute, Brussels, Belgium 2: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Free

University of Brussels, Brussels, Belgium 3: Institute for Frontier Medical Science, Kyoto, Japan The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium which derives from the endoderm and which is surrounded by mesoderm-derived mesenchyme. By morphological analysis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non-polarized epithelial cells. Later, during the second transition the buds reorganize into branched and polarized epithelial monolayers, that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, while its receptor CXCR4, is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analysis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. Interestingly, SDF-1 and CXCR4 are expressed in this organ and AMD3100 treatment of submandibular glands blocks its branching morphogenesis. Alltogether, these data suggest that SDF-1 controls development of several exocrine tissues.

BSCDB & FWO & FEDRA – March 2009 - Leuven Hick Anne-Christine "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 27 Janssens Els


DEVELOPMENTAL RETINOTECTAL AXON PATHFINDING IN ZEBRAFISH: A ROLE FOR MATRIX METALLOPROTEINASES

Els Janssens and Lieve Moons

Research Group Neural Circuit Development and Regeneration Dept. of Biology, Naamsestraat 61,K.U.Leuven, B-3000 Leuven, Belgium

Correct wiring of neuronal circuits in the developing brain relies on the precise spatial positioning of neurons and their axons. The optic circuit, which contains the axons of retinal ganglion cells (RGCs), is a powerful model system to study axon guidance, midline crossing and formation of topographic neuronal maps. Recent loss/gain-of-function studies in different animal models revealed several attractive and repulsive guidance cues that regulate the proper formation of the visual pathway. Recently matrix metalloproteinases (MMPs) have been shown to regulate migration and survival of neurons, axon guidance and myelination in the developing brain. There is also substantial evidence that MMPs participate in the development of retinotectal projections. However, the nature and working mechanisms of these MMPs/TIMPs (tissue inhibitors of metalloproteinases) in retinotectal pathfinding remain completely unidentified. We characterized the expression of several MMPs and TIMPs, identified in zebrafish, in the brain of zebrafish embryos at various time points during development of the retinotectal system by in situ hybridization. Especially zMMP2, zMMP14a, zMMP14b and zTIMP2 show an extensive expression pattern in the brain. Furthermore by using a broad-spectrum MMP inhibitor in combination with DiI (3,3'-dioctadecyloxacarbocyanin perchlorate) injections, we were able to show the involvement of MMPs in axon growth and/or guidance in the retinotectal system of zebrafish. Indeed, our results indicate a severe disruption of axon pathfinding after general MMP inhibition. Altogether, these novel findings reveal that MMPs are expressed in the zebrafish brain during development of the visual pathways and that axon pathfinding in the retinotectal system of zebrafish can be disrupted by blockade of MMP signaling. Our results might have implications for neural circuit development and regeneration.

BSCDB & FWO & FEDRA – March 2009 - Leuven Janssens Els "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 28 Janssens Sylvie


IDENTIFICATION AND CHARACTERIZATION OF NEW WNT-betaCATENIN TARGET GENES IN XENOPUS

Sylvie Janssens (1,2), Tinneke Denayer (1,2), Tom Deroo (1,2) and Kris Vleminckx (1,2)

1: Department for Molecular Biomedical Research, VIB 2: Department of Biomedical Molecular Biology, Ghent University Technologiepark 927, B-9052 Ghent, Belgium The Wnt/beta-catenin pathway has a pivotal role during animal development and in adult life. Its activation leads at its nuclear endpoint to the transcriptional activation of specific target genes. In many different species and systems putative Wnt target genes have been identified. Still, only a limited number are verified to be directly regulated by the pathway. Since they represent potential therapeutic anchor points for the treatment of cancer or for stem cell therapies, there is a tremendous need for the identification of new primary Wnt target genes that are only induced in a particular cellular context. For this they are ideally identified in the complex context of a whole embryo, which provides tissue- and organ-specificity, and with an inducible system, which favors the identification of true primary targets. Hence, we designed and optimized inducible transgenic Wnt pathway interfering (activating or repressing) systems acting at the nuclear endpoint of the pathway and performed microarray analysis on neurula stage Xenopus embryos. Interestingly this generated a list of less than 100 genes which were affected by manipulating the Wnt pathway. Remarkably, 21% of these were known Wnt target genes. Several others were genes associated with processes where the canonical Wnt pathway is known to be involved at the stage analyzed (e.g. anterior-posterior patterning and differentiation) further validating our system. Analysis of these genes by in situ hybridization showed that several known and novel genes were induced by the ubiquitous activation of the Wnt pathway, however, the expression of these genes was mostly not observed outside their normal expression domain (e.g. tissue or organ). This means that these genes are under strict epigenetic regulation or require the input of other signals that are specific for the cellular environment. We are now exploiting this remarkable feature to identify novel organ-specific Wnt/beta-catenin target genes.

BSCDB & FWO & FEDRA – March 2009 - Leuven Janssens Sylvie "CELL FATE DETERMINATION IN THE EMBRYO"

Poster 29 Kahr Irene


FUNCTIONAL ANALYSIS OF THE CANDIDATE TUMOR SUPPRESSOR PROTEIN PROTOCADHERIN-10

Irene Kahr (1,2), Uta Fuchs (1,2), Katrien Staes (1,2) and Frans van Roy (1,2)

1: Department for Molecular Biomedical Research, VIB 2: Department of Biomedical Molecular Biology, Ghent University Technologiepark 927, B-9052 Ghent, Belgium Protocadherins constitute the largest subgroup in the cadherin superfamily of adhesion molecules. They have six or seven cadherin motifs tandemly repeated in their extracellular domain, but their cytoplasmic regions have no similarity to those of classical cadherins and are also very distinct from each other, suggesting a capacity for interaction with various signaling pathways and/or cytoskeletal elements. It has been shown that protocadherin-10 (Pcdh10) mediates homophilic cell adhesion in vitro, although the binding is weaker than that by classical cadherins, and cell sorting in cell culture and in vivo (Hirano et al., 1999). Pcdh10 has been implicated in cancer, as multiple human carcinoma and lymphoma cell lines as well as tumors show frequent promoter methylation of PCDH10. Ectopic expression of PCDH10 in tumor cells with methylated PCDH10 promoter strongly suppressed tumor cell growth, migration and invasion (Ying et al., 2006 and 2007). We are currently generating conditional Pcdh10 KO mouse models to delete Pcdh10 in a tissue- and time-specific manner. On the one hand, a model in which all isoforms of Pcdh10 can be knocked out will be established. This mouse will then be crossed with different Cre mice as well as various tumor mouse models to elucidate the role of Pcdh10 in several cellular processes. Additionally, a second mouse model is generated, in which only the long isoforms are conditionally knocked out, what includes the ablation of two conserved cytoplasmic domains, CM1 and CM2. The latter mouse model will be used to explore the role of these conserved domains in various intracellular signaling pathways. To identify new cytoplasmic interaction partners of PCDH10, we initiated the search for putative interactors using an innovative technology called MAmmalian Protein-Protein Interaction Trap (MAPPIT) (Eyckerman et al., 2001). The first MAPPIT screen (Lievens et al., 2004) for interaction partners of the cytoplasmic domain of PCDH10 has been completed and the results were analyzed in cooperation with the group of Prof. Jan Tavernier, Ghent University. In addition, Pcdh10 was used in a commercial Yeast 2-Hybrid (Y2H) screen (Hybrigenics). In both assays until now only a few weak interactors were identified. More recently, a Y2H screen

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Spring Meeting of the BSCDB, jointly organized with FWO-...(PCP) genes celsr2/off-road and frizzled3a/off-limits block FBMN caudal migration. Here, we investigated the role of cadherins