Understanding the Role of Prdm12b in Zebrafish Development

Abstract

....................................................................................................... IV LIST OF FIGURES .............................................................................................. X LIST OF TABLES .............................................................................................. XII LIST OF VIDEOS .............................................................................................. XIII CHAPTER I: INTRODUCTION .............................................................................1 VERTEBRATE CENTRAL NERVOUS SYSTEM DEVELOPMENT ...................2 The neural plate formation .............................................................................2 Transformation of neural plate into neural tube ..............................................5 Neural tube patterning....................................................................................6 PRDM FAMILY MEMBERS .............................................................................15 PRDMs as gene expression regulators ........................................................16 Role of PRDMs in tissue specification and differentiation ............................17 PRDMs in nervous system ...........................................................................20 ZEBRAFISH LOCOMOTION CIRCUITRY ......................................................24 Spinal cord neurons .....................................................................................24 Motor behaviors in zebrafish ........................................................................27 CONTRIBUTION OF THIS WORK TO THE FIELD .........................................31 VII CHAPTER II: ZEBRAFISH PRDM12B ACTS INDEPENDENTLY OF NKX6.1 REPRESSION TO PROMOTE ENG1B EXPRESSION IN THE NEURAL TUBE P1 DOMAIN ........................................................................................................33 BACKGROUND ...............................................................................................34 METHODS ......................................................................................................37 Zebrafish care ..............................................................................................37 Generation of CRISPR/Cas9 mutant zebrafish lines ....................................37 Antisense morpholino oligonucleotide injections ..........................................40 In situ RNA hybridization ..............................................................................40 Luciferase reporter assays ...........................................................................41 Co-immunoprecipitation and Western blotting .............................................42 Immunocytochemistry ..................................................................................42 Behavioral analysis ......................................................................................43 Genotyping ...................................................................................................43 RESULTS ........................................................................................................44 Germ line disruption of prdm12b blocks eng1b expression in the p1 domain .....................................................................................................................44 The defective escape response in prdm12b mutants is caused by disruption of the Mauthner cell pathway .......................................................................58 Prdm12b acts as a repressor in vitro............................................................66 Prdm12b interacts with the Bhlhe22 transcription factor and the EHMT2 methyltransferase.........................................................................................72 VIII bhlhe22 is not required for eng1b expression in the zebrafish p1 domain ...76 prdm12b does not maintain the p1 domain by repressing nkx6.1 ................81 DISCUSSION ..................................................................................................91 prdm12b germ line mutants recapitulate the phenotype observed using antisense-based approaches .......................................................................92 prdm12b is a bona fide transcriptional repressor .........................................93 An undefined domain persists at the p1 position in prdm12b mutants .........95 CONCLUSION.................................................................................................97 AUTHORS CONTRIBUTIONS ........................................................................97 ACKNOWLEDGEMENTS ................................................................................98 CHAPTER III: DISCUSSION ..............................................................................99 CORRELATION BETWEEN MORPHOLINO-INDUCED AND MUTANT PHENOTYPES IN PRDM12B LOCUS .......................................................... 101 THE FATE OF THE REMAINING P1 DOMAIN IN PRDM12B MUTANTS IS UNDEFINED ................................................................................................. 102 THE ROLE OF PRDM12B IN DORSOVENTRAL PATTERNING IS COMPLEX ...................................................................................................................... 105 Further in vivo analysis of Prdm12b interaction partners namely Bhlhe22 and EHMT2/G9a is required ............................................................................. 105 What are the regulatory relationships of a subset of genes in the p0-p2?.. 109 IX THE RELATIONSHIP OF GENE EXPRESSION THROUGH THE P0-P2 DOMAIN MARKERS DOES NOT INVOLVE SIMPLE DIRECT INTERACTIONS ............................................................................................ 112 REMAINING QUESTIONS AND FUTURE DIRECTIONS OF THIS WORK .. 115 What are the prdm12b direct targets? ........................................................ 115 Do p1-derived neurons restrict Mauthner cells to fire only once? .............. 117 CONCLUSION............................................................................................... 120 APPENDIX: THE EFFECT OF PRDM12B AND BHLHE22 MISEXPRESSION .......................................................................................................................... 122 INTRODUCTION ........................................................................................... 123 METHODS .................................................................................................... 123 Zebrafish care ............................................................................................ 123 Zebrafish were handled as discussed in Chapter II. .................................. 123 Zebrafish embryonic injections ................................................................... 123 In situ RNA hybridization ............................................................................ 124 RESULTS ...................................................................................................... 125 Gain of function prdm12b and/or bhlhe22 does not affect eng1b expression ................................................................................................................... 125 DISCUSSION ................................................................................................ 128 REFERENCES.................................................................................................. 129