SUBLINEAGE-SPECIFIC CUES REQUIRED FOR EARLY AND LATER NEURAL CREST DEVELOPMENT IN THE ZEBRAFISH, DANIO RERIO DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brigitte Louise Arduini, B.S. ***** The Ohio State University 2005 Dissertation Committee: Approved by Dr. Paul D. Henion, Advisor Dr. Helen Chamberlin Advisor Graduate Program in Molecular Genetics Dr. Mark Seeger Dr. Harald Vaessin ABSTRACT The neural crest (NC) of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The generation of NC-derived cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. Zebrafish trpm7/touchtone, endzone, and foxd3/sympathetic mutation1 are discrete loci required by subsets of neural crest derivatives. Severe mutant alleles of the divalent cation channel gene trpm7 are lethal and cell-autonomously cause reductions in the number and size of crest-derived melanophores, while sparing other NC lineages. The deficit in cell numbers can be accounted for at least in part by cell death of melanophore precursors. Pleiotropic effects in non-crest derived tissues, including altered order of bone ossification and kidney dysfunction, are observed in homozygotes for semi-viable alleles. Mutations in endzone affect all three pigment cell lineages found in zebrafish. Normally large and stellate, melanophores and xanthophores take on a rounded, punctate appearance in these mutants. Iridiphores are also reduced in size. These three cell types appear to be similarly reduced in numbers in endzone mutant embryos. While neuronal, glial and ectomesenchymal ii derivatives of the NC appear to be normal in endzone homozygotes, the non- crest-derived pigmented retinal epithelium is developmentally delayed, pointing to pleiotropism for these mutations, as well. Both trpm7 and endzone act relatively late during chromatophore development; accordingly molecular analyses reveal no defects in the early NC cell populations of these mutants. foxd3sym1 affects multiple derivatives within the NC. FoxD3 is required for sympathetic and sensory neuron development, but appears to be dispensable for chromatophore lineages. Anterior elements of the craniofacial skeleton are reduced, and posterior elements are missing, indicating a role for foxd3 in axial patterning of the pharyngeal arches. Expression of critical transcription factors in early crest cells and crest migration are both abrogated, although the premigratory NC population appears to be induced normally. Simultaneous abrogation of Foxd3 and Tfap2α function leads to loss of all chromatophores and most craniofacial cartilages. Collectively, these data suggest that subpopulations with distinct genetic requirements exist within the early neural crest and its later sublineages. iii DEDICATION To Mom, for all that you do. iv ACKNOWLEDGEMENTS I would like to thank Paul Henion for excellent guidance and training, for fresh garden vegetables, and for having a sense of humor. I am grateful that his door was (almost) always open, that he taught when needed, and yet allowed me to find my own way. The importance of the mentor-student relationship cannot be overestimated; I am fortunate to have found this one both very challenging and very rewarding. Thank you also to my lab mates, An Min, Marsha Lucas, Myron Ignatius, Gao Juan, Smitha Malireddy, Roopa Nambiar, Natalie Gentry, Luo Rushu and Matt Frieda, for discussions thoughtful and capricious, and for their friendship. I am appreciative of Christine Beattie and the members of her lab, Louise Rodino- Klapac, Michelle McWhorter, Tessa Carrel, Emily Tansey and Wang Chunping, for sharing expertise, laughter and a penchant for Indian food. I would like to thank Michelle Gray and Anil Challa, who were indispensable to my early training and to my personal growth. To the members of my committee, Helen Chamberlin, Mark Seeger and Amanda Simcox, I am grateful for their highly constructive roles in my scientific development, as well as their significant time and efforts on my behalf. I am also greatly appreciative of Harald Vaessin for stepping in when needed. Thank you v to Heithem El-Hodiri for enthusiasm for science and for people, and for organizing decompression time. Very special thanks to Cliff Gebhardt and Martin Gillard, from whom I first learned to love biology, and to Rhonda Curtis, for so much respect and encouragement. I must also thank the staff of the Molecular Neurobiology Center and the Department of Molecular Genetics, who routinely uncomplicated the practical necessities of science and education. I thank my family for innumerable contributions, my mother Judy Leonhart, my father Bino Arduini, my sister Noelle Arduini, and my grandparents Brigitte Heckmann, Bino and Louise Arduini. Their love, support and help have enabled my achievements at every level. vi VITA March 12, 1977. Born - Oswego, New York May 30, 1999. B.S. Biology Cornell University 1999 - 2000. Teaching Asst. The Ohio State University. 2000 - 2005. Research Asst. The Ohio State University. PUBLICATIONS Research Publications 1. Luo, R., An, M., Arduini, B.L., and Henion, P.D. (2001) Specific pan-neural crest expression of zebrafish crestin throughout embryonic development. Dev Dyn 220(2): 169-174. 2. Arduini, B.L. and Henion, P.D. (2004) Melanogenic sublineage-specific requirement for zebrafish touchtone during neural crest development. Mech Dev 121(11):1353-64. 3. Elizondo, M.R., Arduini, B.L., Paulsen, J., MacDonald, E.L., Sabel, J.L., Henion, P.D., Cornell, R.A, Parichy, D.M. (2005) Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7. Curr Biol 15(7):667-71. FIELDS OF STUDY Major Field: Molecular Genetics vii TABLE OF CONTENTS P a g e Abstract . .ii Dedication . iv Acknowledgements . .v Vita . .vii List of Tables. .x List of Figures . .xi List of Abbreviations. xiv Chapter 1: Introduction. 1 Neural plate border induction . 3 Epithelial-to-mesenchymal transition and neural crest migration. .9 Specification of sublineages within the neural crest. .13 Conclusion. 22 Tables and Figures. .24 Chapter 2: Specific pan-neural crest expression of zebrafish crestin throughout embryonic development. .28 Abstract. .28 Introduction. .29 Results and Discussion . 31 Materials and Methods. .35 Tables and Figures . 37 Chapter 3: Melanogenic sublineage-specific requirement for zebrafish touchtone during neural crest development. 42 Abstract. .42 Introduction. .43 viii Results. .48 Discussion. .58 Materials and Methods. 65 Tables and Figures. 71 Chapter 4: Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7. .81 Abstract. .81 Results and Discussion. 82 Materials and Methods. .87 Tables and Figures. .93 Chapter 5: Zebrafish endzone regulates neural crest-derived chromatophore morphology and differentiation . .106 Abstract. .106 Introduction. .107 Results. 112 Discussion. 121 Materials and Methods. .125 Tables and Figures. .130 Chapter 6: sympathetic mutation 1 encodes zebrafish foxd3 and is differentially required for early neural crest development. 140 Abstract. 140 Introduction. .141 Results. 143 Discussion. 156 Materials and Methods. .163 Tables and Figures. .166 Chapter 7: Genetic interaction of zebrafish foxd3 and tfap2 in neural crest derived pigment and craniofacial development. 181 Abstract. .181 Introduction. .182 Results. 184 Discussion. 189 Materials and Methods. .193 Tables and Figures. .196 Chapter 8: Discussion. 202 Sublineage-specific genetic requirements within the neural crest. 202 Genetic heterogeneity within distinct neural crest sublineages. 205 List of References. 208 ix LIST OF TABLES Table Page 2.1 Molecular markers for neural crest and neural crest-derived cells . 37 3.1 Other neural crest derivatives are normal in tct mutant embryos . 71 3.2 Melanophores are reduced in tct mutants. 72 3.3 Depletion of melanoblasts in tct.