1 Acknowledgments I want to express my sincere gratitude to my advisor, Vladimir Korzh, for accepting me into his laboratory If this postgraduate program would be likened to a journey through an exotic land, then I could not have found a better guide Encyclopedic in knowledge and skilled in techniques, he provided an ideal environment for me to mature into a working scientist I want to thank members of my thesis advisory committee, Dr Thameem Dheen, Dr Suresh Jesuthasan and Prof Wan-Jin Hong for their valuable advice and guidance Dr Dheen has laid much of the foundation for the work described in this thesis Dr Suresh and Prof Hong have influenced the direction of this project more than they realized I thank members of V.K lab.- Alexander Emelyanov, the resident guru in all things molecular who laid the foundation for Fz work and provided me with many of the reagents used in this study; Sergei Parinov, an expert in transposon-mediated enhancer trap, an enthusiastic diver and a fabulous photographer, for encouragement and distraction when I needed it; Michael Richardson, for laying the groundwork on Fz2, and shared my appreciation of wit, understatement and sarcasm; Cathleen Teh, an expert in zebrafish electroporation, for making the BAC-recombinant Fz construct I am also grateful to Shang-Wei Chong and Kar-Lai Poon, fellow travellers in the exotic land, for putting up with me My warmest appreciation for Lee-Thean Chu, another fellow traveller, for painstakingly proof-reading my manuscripts and this thesis I thank Paramjeet Singh, a dear friend and mentor in my formative years, for showing me the meaning of science and how it ought to be conducted I will never forget his advice to ‘Never let work get in the way of progress!’ Many fine ideas were born over coffee or beer with him And many other silly ones were shot down by him even before I lifted a pipette, saving me precious time He showed me that it’s not how much work I do, but how much of that work is absolutely essential to answer the question Most importantly, is it the right question to begin with I thank Chai Chou, fellow lunatic and masochist, for showing me the meaning of staying the course In the face of difficulties bordering on the absurd, he stoically marched on At times when the going got too tough for me, I only have to look at him to renew my strength I have very fond memories of my time in IGBMC (Strasbourg, France) I thank Uwe Strähle, Ferenc Müller, Patrick Blader, Thomas Dickmeis and Nadine Fischer for making me feel at home 2000 km away I also thank Stephen Wilson, Miguel Concha and C.P Heisenberg of the University of College, London (UCL) for their hospitality and kind tutelage in the fine art of single cell transplantation I am also indebted to R Moon, S Vriz and U Takeda for reagents and cDNAs To Bill Chia and Xiao-Hang Yang for discussions during the initial stages of this work To Chin-Heng, Amy and everyone in the fish facility; without them there would be no work I would also like to thank many of my colleagues in Temasek Life-Science Laboratory (formally Institute of Molecular Agrobiology) for support and encouragement This work was made possible by the generous support from the Agency for Science, Technology and Research of Singapore Finally, to Kalwant Singh (1960-1992) He’s got a Ticket To Heaven This work would not be complete without the generous contributions from Sasha, Cathleen and Mike Thank you all, from my heart Table of Contents Acknowledgments Summary Publications Chapter 1: Introduction Chapter 2: Wnt signalling mediated by Tbx2b is required for cell migration during formation of the neural plate 25 2.1: Results 26 2.2: Discussion 50 Chapter 3: Zebrafish Frizzled is required for dorsoventral specification in the gastrula leading to the formation of posterior structures 60 3.1: Results 61 3.2: Discussion 70 Conclusions 74 Appendix 79 Experimental Procedures 86 References 99 List of Figures Fig Phenotypes of tbx2b morphants at 24 hpf 27 Fig shh expression in control and tbx2bMO injected embryo 29 Fig 3A Anti-tbx2bMO blocks transcription of tbx2b mRNA and is distributed unevenly in the blastula 31 Fig 3B Anti-tbx2bMO impairs morphogenesis of anterior forebrain 32 Fig Tbx2b is required for cells to adopt a neural fate 34 Fig Tbx2b is required for convergence cell movement 35 Fig ‘Exclusion’ phenotype can be rescued by tbx2b mRNA 38 Fig Inhibition of Fz7 signalling leads to the ‘exclusion’ phenotype 40 Fig Tbx2b is regulated by Fz7 42 Fig Early function of Tbx2b and Fz7 is required for cell adhesion 46 Fig 10 Cell movement during gastrulation is affected in knockdown cells 49 Fig 11 Early expression pattern of fz2 63 Fig 12 Fz2 function is required to promote neural fates 65 Fig 13 Transplanted deep cells move to ventral regions 67 Fig 14 Fz2 is necessary for formation of posterior structures 69 Fig 15 Regulation of shh promoter and enhancer elements in COS7 and HeLa cells 85 Summary During gastrulation, optimal adhesion and receptivity to signalling cues are essential for cells to acquire new positions and identities via coordinated cell movements T-box transcription factors and the Wnt signalling pathways play important roles in these processes Here we show that Tbx2b is required cell-autonomously for cell adhesion and cell movement in the embryonic ectoderm In chimeric embryos, cells deficient in Tbx2b are defective in cell adhesion and fail to migrate to the neural plate Using this ‘exclusion’ phenotype as a screen, we show that Tbx2b acts within the context of Fz7 via components of the canonical Wnt pathway Independent of cell movement, Tbx2b is also required for neuronal differentiation In contrast to studies in amniotes, our screen failed to demonstrate a role for FGF signalling in the dorsal movement of embryonic ectoderm leading to neural plate formation Instead, our results illustrate the importance of Tbx2b-mediated Wnt signalling in this process We have also fate-mapped a previously undefined population of deep lying vegetal cells in the zebrafish blastula, and showed that Fz2 function is essential for their migration to the ventral side of the gastrula and their subsequent incorporation into the posterior structures of the embryo This result highlights the early commitment of cell fate during the blastula period, and links the positions of cells in the gastrula to their earlier positions in the blastula Thus, Fz2 is required for the initial specification of the dorsoventral axis in the gastrula We further demonstrate that Fz2 is required for the specification of posterior paraxial mesoderm Publications Steven Fong, Alexander Emelyanov, Cathleen Teh and Vladimir Korzh Wnt signaling mediated by Tbx2b regulates cell migration during formation of the neural plate (Development, submitted, joint first authorship with Emelyanov) Sudha Puttur Mudumana, Thomas Dickmeis, Steven Fong, Inna SleptsovaFriedrich, Alexander Emelyanov, Zhiyuan Gong, Uwe Strähle, Vladimir Korzh colIXa2 and determination of lineage of non-notochordal precursors of axial structures of vertebrates (Development, in preparation) Alexander Emelyanov, Steven Fong, Cathleen Teh, Chen Sok Lam, Vladimir Korzh Zath3 acts as a proneural determinant in multipotential neural precursors in hindbrain (Development, in preparation) Michael Richardson, Steven Fong, Dmitry Bessarab, Alexander Emelyanov, Vladimir Korzh Frizzled2 acts downstream of Wnt3a as a determinant of lateral mesoderm (in preparation, joint first authorship) Lee Thean Chu, Steven Fong, Dmitry Bessarab, Alexander Emelyanov, Suresh Jesuthasan, Andrej Minin and Vladimir Korzh Blastocystic cavity plays a role in establishing first cell lineages in zebrafish (Developmental Biology, in preparation) Chapter 1: Introduction 1.1 Zebrafish as a model organism Zebrafish (Danio rerio) has become an important model organism in the study of vertebrate developmental biology Its main advantage is the availability of numerous genetic mutants (Development Vol 123, 1996) and the relative ease with which mutagenesis can be effected by chemical (ENU), physical (X-ray) and genetic (transposon or viral mediated insertions) means This powerful genetics is not available in Xenopus (although Xenopus tropicalis is currently being developed as a genetic system) and chick Although mice and rats are valuable mammalian genetic systems (natural mutants and ‘knockouts’), the embryos of these animals are not as readily accessible and available as the hundreds of transparent, externally fertilized eggs of the zebrafish Having said this, the mutants currently available for zebrafish are by no means saturating Many genes, when mutated, will lead to very early developmental arrest or death, thus eluding screens designed around observable phenotypic alterations In addition, methods for targeted mutagenesis (similar to ‘knockout’ mice) are currently unavailable in zebrafish Thus, cloning of genes from mutants is a labour intensive candidate gene/map-based effort To complicate matters, it is an accepted view that there was a genome duplication event early in the evolution leading to modern teleosts (Wittbrodt et al, 1998; Woods et al, 2000) As such, redundancy in gene function is not uncommon This should in no way be viewed as an indictment against zebrafish as a model organism Its genetics and embryological accessibility, coupled with modern molecular tools - like dominant negative constructs and the recently developed anti-sense morpholino oligonucleotides which work by inhibiting translation of target mRNAs (Nasevicius and Ekker, 2000) - for manipulating gene function, makes for a powerful system to understand higher vertebrate biology This is particularly evident in the study of human diseases, where many zebrafish mutants resemble human clinical disorders (Dooley and Zon, 2000) 1.2 General zebrafish development The zebrafish embryo develops rapidly at 28°C At 2.75 hours post fertilization (hpf) or midblastula stage (512 cells), zygotic transcription begins This time point marks the midblastula transition (MBT) Cell divisions are very rapid and synchronized in the first 10 cleavages At MBT, the cell cycle begins to lengthen and cells lose their synchrony thereafter At around this time, the first lineage restriction is observed, where the formation of the yolk syncytial layer (YSL) separates the yolk cell from the rest of the blastoderm After one or two more cell divisions, the lineage of epithelial enveloping layer (EVL) cells becomes restricted to exclusively generate the periderm, while the deep layer cells (DEL) proceed to give rise to the embryo proper As such, the YSL is an extra embryonic structure and can thus be seen as the equivalent of the syncytiotrophoblast of the mammalian embryo By 4.5 hpf epiboly movements signal the start of gastrulation Lineage tracing by intracellular injection of dye shows that DEL cells located near the animal pole will give rise to ectodermal fates (including the definitive epidermis) and cells located near the blastoderm margin will give rise to mesodermal and endodermal fates At hpf, during the gastrula period, the dorsal organizer or shield is well defined and gastrulation is now in full swing It is also at this stage that the future fates of respective regions of the gastrula are defined (Kimmel et al, 1990) It has been suggested that the position of a cell in the blastula is unrelated to the position of its descendants in the gastrula due to the extensive cell mixing that occurs 10 during the transition from blastula to gastrula (Warga and Kimmel, 1990) Subsequently, a study demonstrated that the fate of cells could be determined as early as the 16-cell stage (Strehlow et al, 1994) However, due to the extremely large molecular weight of the labelling tracer employed in this study, the finding was controversial It is suggested that the position of blastomeres at the 16-cell stage may indeed predispose them to a particular region of the gastrula, and thus determine their ultimate fates, but the final contribution that any early blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed (Helde et al, 1994; Wilson et al, 1995) By 10 hpf, primary gastrulation is complete At this stage the three germ layers are well defined and the neural plate is formed The first somite appears at about 10.5 hpf and signals the beginning of the segmentation period The tail bud is now formed and with it, the recently proposed secondary gastrulation begins (Kanki and Ho, 1997; Agathon et al, 2003) At 16 hpf (14 somites stage), the notochord separates from the ventral part of the neural keel and the yolk extension begins to protrude At 19 hpf (20 somites stage), the lens placode appears and neurons have growing axons At this stage, trunk myotomes produce weak contractions By 24 hpf, pigments, fins and a beating heart are visible By day 3, the larva breaks free from the chorion which has protected it until now, and becomes free swimming For a thorough review and description of developmental stages, see Kimmel et al, 1995 and Westerfield, 1995 10 106 in embryonic Xenopus ectoderm leads to direct neural induction Genes Dev 9, 2923-2935 He, C., Fong, SH., Yang, D., Wang, GL (1999) BWMK1, a novel MAP kinase induced by fungal infection and mechanical wounding in rice Mol Plant Microbe Interact 12, 1064-1073 Heasman, J (2002) Morpholino oligos: Making sense of Antisense? Dev Biol 243, 209-214 Heisenberg, CP et al (2000) Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation Nature 405, 76-81 Herrmann, BG et al (1990) Cloning of the T gene required in mesoderm formation in the mouse Nature 343, 617-622 Helde, KA., Wilson, ET., Cretekos, CJ and Grunwald, DJ (1994) Contribution of early cells to the fate map of the zebrafish gastrula Science 265, 517-520 Ho, RK and Kane, DA (1990) Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors Nature 348, 728-30 Ho, RK and Kimmel, CB (1993) Commitment of cell fate in the early zebrafish embryo Science 261: 109-111 106 107 Hsieh, JC., Rattner, A., Smallwood, PM and Nathans, J (1999) Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein Proc Natl Acad Sci USA 96, 3546-3551 Jacobs, JJ., Keblusek, P., Robanus-Maandag, E., Kristel, P., Lingbeek, M., Nederlof, PM., van Welsem, T., van de Vijver, MJ., Koh, EY., Daley, GQ and van Lohuizen M (2000) Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers Nat Genet 26, 291-299 Jesuthasan, S and Strähle, U (1997) Dynamic microtubules and specification of the zebrafish embryonic axis Curr Biol 7, 31-42 Jones, CM and Smith, JC (1998) Establishment of a BMP-4 morphogen gradient by long-range inhibition Dev Biol 194, 12-17 Kanki, JP and Ho, RK (1997) The development of the posterior body in zebrafish Development 124, 881-893 Kelly, GM., Erezyilmaz, DF and Moon, RT (1995) Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of betacatenin Mech Dev 53, 261-273 Kim, CH et al (2000) Repressor activity of Headless/Tcf3 is essential for vertebrate head formation Nature 407, 913-916 107 108 Kimmel, CB., Warga, RM and Schilling, TM (1990) Origin and organization of the zebrafish fate map Development 108, 581-594 Kimmel, CB., Warga, RM and Kane, DA (1994) Cell cycles and clonal strings during formation of the zebrafish central nervous system Development 120, 265-276 Kimmel, CB et al (1995) Stages of embryonic development of the xebrafish Dev Dyn 203, 253-310 Kopp, A and Duncan, I (1997) Control of cell fate and polarity in the adult abdominal segments of Drosophila by optomotor-blind Development 124,3715-3726 Korinek, V et al (1997) Constitutive transcriptional activation by a betacatenin-Tcf complex in APC-/- colon carcinoma Science 275, 1784-1787 Korswagen, HC (2002) Canonical and non-canonical Wnt signaling pathways in Caenorhabditis elegans: variations on a common signaling theme Bioessays 24, 801-810 Kudoh, T., Concha, M., Houart, C., Dawid, I and Wilson, S (2004) Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains Development 131, 3581-3592 Kuhl, M et al (2001) Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca(2+) signaling Mech 108 109 Dev 106, 61-76 Kwan, KM and Kirschner, MW (2003) Xbra functions as a swith between cell migration and convergent extention in the Xenopus gastrula Development 130, 1961-1972 Lamb, TM and Harland, RM (1995) Fibroblast Growth Factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern Development 121, 3627-3636 Launay, C., Fromentoux, V., Shi, DL and Boucaut, JC (1996) A truncated FGF receptor blockes neural induction by endogenous Xenopus inducers Development 122, 869-880 Lekven, AC., Thorpe, CJ., Waxman, JS and Moon, RT (2001) Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning Dev Cell 1, 103-114 Lele, Z., Folchert, A., Concha, M., Rauch, GJ., Geisler, R., Rosa, F., Wilson, SW., Hammerschmidt, M., Bally-Cuif, L (2002) parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube Development 129, 3281-3294 Leung, T., Bischof, J., Soll, I., Niessing, D., Zhang, D., Ma, J., Jackle, H., Driever, W (2003) bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression Development 130, 3639-3649 109 110 Lingbeek, ME., Jacobs, JJ and van Lohuizen, M (2002) The T-box repressors TBX2 and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-si ête in the initiator J Biol Chem 277, 2612026127 Liu, P., Wakamiya, M., Shea, MJ., Albrecht, U., Behringer, RR and Bradley, A (1999) Requirement for Wnt3 in vertebrate axis formation Nat Genet 22, 361-365 Luo, W and Lin, SC (2004) Axin: a master scaffold for multiple signaling pathways Neurosignals 13, 99-113 Martinez-Barbera, JP., Toresson, H., Da Rocha, S and Krauss, S (1997) Cloning and expression of three members of the zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4 Gene 198, 53-59 McGrew, LL., Hoppler, S., Moon, RT (1997) Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus Mech Dev 69, 105-114 McGrew, LL., Lai, CJ., Moon, RT (1999) Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistatin Dev Biol 172, 337-342 Miller, JR., Rowning, BA., Larabell, CA., Yang-Snyder, JA., Bates, RL and Moon, RT (1999) Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent 110 111 on cortical rotation J Cell Biol 146, 427-437 Moens, CB and Fritz, A (1999) Techniques in neural development Met Cell Biol 59, 253-272 Moon, RT., Brown, JD and Torres, M (1997) WNTs modulate cell fate and behaviour during vertebrate development Trend Genet 13, 157-162 Moon, RT., Bowerman, B., Boutros, M and Perrimon, N (2002) The promise and perils of Wnt signaling through beta-catenin Science 296, 1644-1646 Moorman, AF., Houweling, AC., de Boer, PA and Christoffels, VM (2001) Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol J Histochem Cytochem 49, 1-8 Muller, CW and Herrmann, BG (1997) Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor Nature 389, 884888 Müller, F., Chang, BE., Albert, S., Fischer, N Tora, L and Strähle, U (1999) Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord Development 126, 2103-2116 Munoz-Sanjuan, I and Brivanlou, AH (2002) Neural induction, the default model and embryonic stem cells Nat Rev Neurosci 3, 271-280 111 112 Myers, DC., Sepich, DS and Solnica-Krezel, L (2002) Convergence and extension in vertebrate gastrulae: cell movements according to or in search of identity? Trend Genet 18, 447-455 Narayanan, K., Williamson, R., Zhang, Y., Stewart, AF and Ioannou, PA (1999) Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E coli DH10B using an inducible homologous recombination system Gene Ther 6, 442-447 Nasevicius, A and Ekker, SC (2000) Effective targeted gene 'knockdown' in zebrafish Nat Genet 26, 216-220 Neave, B., Holder, N and Patient, R (1997) A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in the zebrafish Mech Dev 62, 183-195 Nelson, J and Nusse, R (2004) Convergence of Wnt, β-catenin, and cadherin pathways Science 303, 1483-1487 Ober, EA and Schulte-Merker, S (1999) Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish Dev Biol 215, 167-181 Packham, EA and Brook, JD (2003) T-box genes in human disorders Hum Mol Genet 12, R37-44 112 113 Papaioannou, VE and Silver, LM (1998) The T-box gene family BioEssays 20, 9-19 Papan, C and Campos-Ortega, J A (1997) A clonal analysis of spinal cord development in the zebrafish Dev Genes Evol 207, 71-81 Parr, BA and McMahon, AP (1995) Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb Nature 374, 350-353 Paxton, C., Zhao, H., Chin, Y., Langner, K and Reecy, J (2002) Murine Tbx2 contains domains that activate and repress gene transcription Gene 283, 117-124 Pflugfelder, GO Roth, H and Poeck, B (1992) A homology domain shared between Drosophila optomotor-blind and mouse Brachyury is involved in DNA binding Biochem Biophys Res Commun 186, 918-925 Piccolo, S., Agius, E., Leyns, L., Bhattacharyya, S., Grunz, H., Bouwmeester, T and De Robertis, E.M (1999) The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals Nature 397, 707710 Prince, S., Carreira, S., Vance, KW., Abrahams, A., Goding, CR (2004) Tbx2 directly represses the expression of the p21 WAF1 cyclin-dependent kinase Inhibitor Cancer Res 64, 1669–1674 Rauch, GJ., Hammerschmidt, M., Blader, P., Schauerte, H.E., Strahle, U., Ingham, P.W., McMahon, AP and Haffter, P (1997) Wnt5 is required for tail formation in the zebrafish embryo Cold Spring Harb Symp Quant Biol 62, 113 114 227-234 Roy, S., Wolff, C and Ingham, P (2001) The u-boot mutation identifies a Hedgehog-regulated myogenic switch for fiber-type diversification in the xebrafish embryo Gene Dev 15, 1563-1576 Rulifson, EJ., Wum, CH and Nusse, R (2000) Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless Mol Cell 6, 117-126 Sakaguchi, T., Mizuno, T and Takeda, H (2002) Pattern formation in zebrafish: Results and problems in cell differentiation p1-14 Ed SolnicaKrezel, L Springer Sampath K, Rubinstein AL, Cheng AM, Liang JO, Fekany K, Solnica-Krezel L, Korzh V, Halpern ME, Wright CV (1998) Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling Nature 395, 185-189 Schulte-Merker, S and Smith, JC (1995) Mesoderm formation in response to Brachyury requires FGF signalling Curr Biol 5, 62-67 Shih, J and Keller, R (1994) Gastrulation in Xenopus laevis: involution - a current view Semin Dev Biol 5, 85-90 Showell, C., Binder, O and Conlon, F (2004) T-box genes in early embryogenesis Dev Dyn 229, 201-218 114 115 Sinclair, CS., Adem, C., Naderi, A., Soderberg, CL., Johnson, M., Wu, K., Wadum, L., Couch, VL., Sellers, TA., Schaid, D., Slezak, J., Fredericksen, Z., Ingle, JN., Hartmann, L., Jenkins, RB and Couch FJ (2002) TBX2 is preferentially amplified in BRCA1- and BRCA2-related breast tumors Cancer Res 62, 3587-3591 Sinha, S., Abraham, S., Gronostajski, RM and Campbell, CE (2000) Differential DNA binding and transcription modulation by three T-box proteins, T, TBX1 and TBX2 Gene 258, 15-29 Slusarski, DC., Corces, VG and Moon, RT (1997) Interaction of Wnt and a Frizzled homologue triggers G-Protein-linked phosphatidylinositol signalling Nature 390, 410-413 Smith, J (1999) T-box genes: what they and how they it Trends Genet 15, 154-158 Sokol, S.Y (1996) Analysis of Dishevelled signalling pathway during Xenopus development Curr Biol 6, 1457-1467 Spemann, H (1938) Embryonic development and induction Yale Univ Press, New Haven Spemann, H., and Mangold, H (1924) Über inducktion von embrryonanlagen durch implantation artfremder organisatoren Wilhelm Roux Arch Entw Mech Organ 100, 599-638 115 116 Stachel, SE., Grunwald, DJ and Myers, PZ (1993) Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish Dev 117, 1261-1274 Steinberg, MS and Takeichi, M (1994) Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression Proc Natl Acad Sci USA 91, 206-209 Strehlow, D., Heinrich, G and Gilbert, W (1994) The fates of the blastomeres of the 16-cell zebrafish embryo Development 120, 1791-1798 Streit, A., Berliner, AJ., Papayanoutou, C., Sirulnik, A and Stern, CD (2000) Initiation of neural induction by FGF signalling before gastrulation Nature 406, 74-78 Sumanas, S., Strege, P., Heasman, J., Ekker, SC (2000) The putative wnt receptor Xenopus frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning Development 127, 1981-1990 Sumanas, S., Kim, HJ., Hermanson, S and Ekker, SC (2001) Zebrafish frizzled-2 morphant displays defects in body axis elongation Genesis 30, 114118 Suzuki, A., Thies, RS., Yamaji, N., Song, JJ., Wozney, JM., Murakami, K and Ueno N (1994) A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo Proc Natl Acad Sci USA 91,10255-10259 116 117 Suzuki, T., Takeuchi, J., Koshiba-Takeuchi, K and Ogura, T (2004) Tbx Genes Specify Posterior Digit Identity through Shh and BMP Signaling Dev Cell 6, 43-53 Tada, M and Smith, JC (2000) Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway Development 127, 2227-2238 Tada, M and Smith, JC (2001) T-targets: clues to understanding the functions of T-box proteins Dev Growth Differ 43, 1-11 Takada, S., Stark, KL., Shea, M.J., Vassileva, G., McMahon, JA and McMahon, AP (1994) Wnt-3a regulates somite and tailbud formation in the mouse embryo Genes Dev 8, 174-189 Takabatake, Y., Takabatake, T., Sasagawa, S and Takeshima, K (2002) Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development Dev Growth Differ 44, 257-271 Topczewski, J et al (2001) The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension Dev Cell 1, 251-264 Veeman, MT., Axelrod, JD and Moon, RT (2003) A second canon: Functions and mechanisms of b-catenin-independent Wnt Signaling Dev Cell 5, 367-377 117 118 Vriz, S and Lovell-Badge, R (1995) The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain Gene 153, 275-276 Wallingford, JB., Rowning, BA., Vogeli, KM., Rothbacher, U., Fraser, SE., Harland, RM (2000) Dishevelled controls cell polarity during Xenopus gastrulation Nature 405, 81-85 Wang, Y., Huso, D., Cahill, H., Ryugo, D and Nathans, J (2001) Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene J Neurosci 21, 4761-4771 Warga RM, Kimmel CB (1990) Cell movements during epiboly and gastrulation in zebrafish Development 108, 569-580 Wessely, O et al (2001) Neural induction in the absence of mesoderm: bcetenin-dependent expression of secreted BMP antagonists at the blastula stage in Xenopus Dev Biol 234, 161-173 Westerfield, M (1995) Editor, The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio) 3rd Edition Wharton, KA (2003) Runnin’ with the Dvl: Proteins that associate with Dsh/Dvl and their significance in Wnt signal transduction Dev Biol 253, 1-17 Wilson, ET., Cretekos, CJ., Helde, KA (1995) Cell mixing during early epiboly in the zebrafish embryo Dev Genet 17, 6-15 118 119 Wilson, PA and Hemmati-Brivanlou, A (1995) Induction of epidermis and inhibition of neural fate by Bmp-4 Nature 376, 331-333 Wilson, SI and Edlund, T (2001) Neural induction: toward a unifying mechanism Nat Neurosci 4, 1161-1168 Wilson, V and Conlon, F (2002) The T-Box family Genome Biol 3: 3008.13008.7 Wilson, V., Manson, L., Skarnes, WC and Beddington, RSP (1995) The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation Development 121, 877-886 Winklbauer, R., Medina, A., Swain, RK., Steinbeisser, H 2001 Frizzled-7 signalling controls tissue separation during Xenopus gastrulation Nature 413, 856-860 Wittbrodt, J., Meyers, A and Schartl, M (1998) More genes in fish? BioEssays 20, 511-515 Woods, IG., Kelly, PD., Chu, F., Ngo-Hazelett, P., Yan, YL., Huang, H., Postlethwait, JH and Talbot, WS (2000) A comparative map of the zebrafish genome Genome Res 10, 1903-1914 Yanagisawa, KO and Fujimoto, H (1977) Differences in rotation-mediated aggregation between wild-type and homozygous Brachyury (T) cells J Embryol Exp Morphol 40, 277-283 119 120 Yoshikawa, Y., Fujimori, T., McMahon AP, Takada S (1997) Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse Dev Biol 183, 234-242 Zhang, Y., Neo, SY., Han, J and Lin, SC (2000) Dimerization choices control the ability of axin and dishevelled to activate c-Jun N-terminal kinase/stress-activated protein kinase J Biol Chem 275, 25008-25014 120 ... (Dsh) The amino-terminal extracellular Wnt binding domain and the 18 19 carboxy-terminal PDZ-binding domain of the Fz protein are known to be essential for Fz function Deletion of the PDZ-binding... In this thesis, we use the zebrafish as a model to study the molecular mechanisms behind the specification of embryonic ectoderm Specifically, we attempt to isolate the role of Tbx2b during the. .. finally folds into a tube, what was initially the margin of the neural plate now becomes the most dorsal portion of the neural tube (the roof plate) and the epidermis from both halves of the embryo