The role of pou2/spiel-ohne-grenzen (spg) in brain and endoderm development of the zebrafish, Danio rerio D I S S E R T A T I O N zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Dipl. Biol. Gerlinde Reim geboren am 11.Oktober 1970 in Mistelbach, Österreich Gutachter: Prof. Dr. Michael Brand Prof. Dr. Francis Stewart Priv. Doz. Dr. Jochen Wittbrodt Eingereicht am: 6. Juni 2003 Tag der Verteidigung: - “ Der Mensch ist nur da ganz Mensch, wo er spielt ” Friedrich Schiller Acknowledgements At the end of the year 1998, inquiring the possibility to do my PhD in developmental biology, I had a revealing visit in Heidelberg to Michael Brand, pioneering the field of neural development of the zebrafish. His vivid interest in developmental biological questions encouraged me to embark on an adventurous journey into zebrafish development, which was completely new to me at that time: this started to change in spring 1999, after Michael Brand had accepted me to join his laboratory... ...Reaching the finale of this adventurous journey, I am very grateful to Michael Brand for introducing me to developmental aspects of the zebrafish. In particular, I would like to thank Michael Brand for his supervision and continuous support during my thesis. His stimulating and decidedly open-minded discussions deepened my interests for developmental biology and permitted explorations into other than neuro-developmental fields. Starting in Heidelberg and continuing, from 2001 onwards, in Dresden, I had the opportunity to meet and to work with people in Michael Brand’s laboratory who contributed to an excellent working atmosphere. I would like to say “thank you” to all past and present members of the Brand laboratory for helpful experimental advices and sharing interests in biological problems – but also, equally important, for their friendship and interest at the personal level. With the same intention, I would like to thank the members of Carl-Philipp Heisenberg’s laboratory. I thank Florian Raible for various discussions and his unremitting help, but also for musical performances like Brahm’s Hungarian Dances and “unconventional interpretations” of Schubert’s piano pieces. I would like to thank Chris Klisa for identifying and providing me with “spiel-ohne-grenzen” mutant carriers, more than that, for being an appreciated companion, especially in playing the piano. I am also grateful to Chris and Florian for proofreading of this thesis. Moreover, I thank Evelyn Lehman and Günter Junghanns for their excellent maintenance of the fish facility, and Shawn Burgess as well as Yutaka Kikuchi for their collaborations and kind support with materials. I am grateful to Dr. Jochen Wittbrodt and Prof. Dr. Francis Stewart for reviewing my thesis. Finally, I would like to thank my mother Ernestine and my sister Eva Maria, and last, but not least – Johannes for supporting me in the course of the last years in manifold instances. Summary The central theme of development, how cells are organized into functional structures and assembled into whole organisms, is addressed by developmental biology. One important feature of embryonic development is pattern formation, which is the generation of a particular arrangement of cells in three-dimensional space at a given point of time. Central to this work is the model system of the zebrafish, Danio rerio. The aim of the first part of this study was to try to understand how a distinct part of the embryonic brain called midbrain-hindbrain boundary (MHB), a region that acts as an organizer for the adjacent brain regions, is established in vertebrates. spiel-ohne-grenzen (spg) is one mutant which interferes with MHB development. Here, I addressed the role of pou2 in brain development by molecular, phenotypical and functional analysis. By genetic complementation and mapping I could elucidate the molecular nature of this mutant and found that the pou2 gene encoding the POU domain transcription factor is affected in spg mutant embryos. By chromosomal syntenic conservation, phylogenetic sequence comparison, and expression and functional data I imply that pou2 is the orthologue of the mammalian Oct4 (Pou5F1) gene. I find by detailed expression and transplantation analysis that pou2 is cell autonomously required within the neuroectoderm to activate genes of the MHB and hindbrain primordium, like pax2.1, wnt1, gbx2 or krox20. By gain-of-function experiments I demonstrate that pou2 synergizes with Fgf8 signaling in order to activate particularly the hindbrain primordium. Since pou2 is already provided to the embryo by the mother, I generated embryos which lack maternal and zygotic pou2 function (MZspg) to reveal a possible earlier than neuroectodermal role of pou2. In the second part of this work I demonstrate that pou2 is a key factor controlling endoderm differentiation. By expression and gain-of-function analysis I suggest a cell autonomous function for Pou2 in the first step of endodermal differentiation. By gain-of-function experiments involving the gene encoding the HMG transcription factor Casanova (Cas) I show that both Cas and Pou2 are necessary to activate expression of the endodermal differentiation marker sox17 in a mutually dependent way, and that the ability of Cas to ectopically induce sox17 strictly requires Pou2. I conclude that both maternal and zygotic pou2 function is necessary for commitment of endodermal progenitor cells to differentiate into endodermal precursor cells. 4 Table of Contents ACKNOWLEDGEMENTS....................................................................................................................................2 SUMMARY........................................................................................................................................................3 TABLE OF CONTENTS ......................................................................................................................................4 INDEX OF FIGURES ..........................................................................................................................................7 ABBREVIATIONS..............................................................................................................................................8 1 INTRODUCTION ...................................................................................................................................9 1.1 DEVELOPMENTAL BIOLOGY AND ITS ELEMENTARY CONCEPTS ......................................................9 1.2 THE ZEBRAFISH: A VERTEBRATE SYSTEM TO STUDY ONTOGENETIC DEVELOPMENT ...................10 1.3 ONTOGENETIC DEVELOPMENT OF THE ZEBRAFISH........................................................................12 1.4 THE IDENTIFICATION OF GENES WITH ESSENTIAL FUNCTIONS IN ZEBRAFISH DEVELOPMENT......16 1.5 POU DOMAIN TRANSCRIPTION FACTORS REGULATE GENE EXPRESSION DURING DEVELOPMENT ... ........................................................................................................................................................18 1.6 EMBRYONIC DEVELOPMENT OF THE VERTEBRATE BRAIN.............................................................21 1.7 THE ROLE OF THE MIDBRAIN-HINDBRAIN (MHB) ORGANIZER .....................................................23 1.8 THE ROLE OF POU2 (SPG) IN MHB ORGANIZER DEVELOPMENT ...................................................24 1.9 EARLY DEVELOPMENT OF ENDODERM FORMATION ......................................................................25 1.10 THE ROLE OF POU2 IN ENDODERM DEVELOPMENT........................................................................28 1.11 QUESTIONS AND AIMS OF THIS THESIS...........................................................................................29 2 RESULTS ...............................................................................................................................................30 2.1 MOLECULAR CHARACTERIZATION OF SPIEL-OHNE-GRENZEN (SPG)..............................................30 2.1.1 Mutagenesis screens yield new alleles of spiel-ohne-grenzen (spg)..................................30 2.1.2 The zebrafish pou2 gene is mutated in spg.........................................................................30 2.1.3 Autoregulation of pou2 suggests spghi349 as the strongest spg allele.................................33 2.1.4 The zebrafish pou2 is the orthologue of the mouse Oct4...................................................33 2.2 THE ROLE OF SPG/POU2 IN NEUROECTODERMAL DEVELOPMENT..................................................34 2.2.1 spiel-ohne-grenzen (spg) is required for midbrain-hindbrain boundary and hindbrain development .........................................................................................................................34 2.2.2 How can the spg MHB phenotype be related to pou2’s function ? ...................................36 2.2.3 Establishment and maintenance of the MHB is affected in spg mutants...........................38 2.2.4 Positioning of the MHB is normal in spg embryos.............................................................40 2.2.5 Early failure of hindbrain gene expression in spg mutants ...............................................42 2.2.6 Prosencephalic gene expression is caudally expanded in spg...........................................44 2.2.7 Mesendoderm development in spg embryos .......................................................................47