The Establishment and Regulation of Melanocyte Stem Cells in Zebrafish

Stem cells are the cells that regulate the growth and repair of tissues in adult organisms. In this thesis, I sought to develop methodologies to dissect the function and regulation of stem cells during zebrafish melanocyte regeneration. In the first part of this thesis, I develop a drug based method of ablating melanocytes in the adult zebrafish body. The drug is a copper chelator, neocuproine: NCP) that I show causes death specifically of melanocytes in adult, which allows for regeneration from melanocyte stem cells: MSCs). In the next part of the thesis, I employ clonal lineage statistical analyses to study the establishment, recruitment, and proliferation, differentiation, and survival of MSC daughter cells during larval melanocyte regeneration. These analyses suggest that MSCs are likely recruited at random for each regeneration event, and that approximately 84% of MSCs are recruited for any regeneration event. I demonstrate that kit signaling has a greater requirement during larval regeneration than during ontogeny and compare the regeneration of kit heterozygotes to wild type. The mutant heterozygotes have normal MSC recruitment and normal proliferation, differentiation, and survival of daughter cells. The mutant has defective MSC establishment, with this defect being quantitatively sufficient to explain the regeneration defect observed. I then used further clonal lineage analysis to suggest that reduction of kit signaling causes inappropriate differentiation of fated MSCs into ontogenetic melanocytes. These analyses are not unique for comparison of kit mutants to wild type, so can easily be applied to dissect any gene or drug which affects regeneration. In the final part of the thesis, I explore how many spermatagonia form the adult zebrafish male germline. An understanding of this number allows for efficient mutant screens, an essential part of the genetic dissection of any process. The zebrafish has approximately 485 spermatagonia, giving each male approximately 970 genomes which can be mutagenized. This number can be considered during mutant screen designs to eliminate redundant screening

Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue

As forward genetic screens in zebrafish become more common, the number of mutants that cannot be identified by gross morphology or through transgenic approaches, such as many nervous system defects, has also increased. Screening for these difficult-to-visualize phenotypes demands techniques such as whole-mount in situ hybridization (WISH) or antibody staining, which require tissue fixation. To date, fixed tissue has not been amenable for generating libraries for whole genome sequencing (WGS). Here, we describe a method for using genomic DNA from fixed tissue and a bioinformatics suite for WGS-based mapping of zebrafish mutants. We tested our protocol using two known zebrafish mutant alleles, gpr126st49 and egr2bfh227, both of which cause myelin defects. As further proof of concept we mapped a novel mutation, stl64, identified in a zebrafish WISH screen for myelination defects. We linked stl64 to chromosome 1 and identified a candidate nonsense mutation in the F-box and WD repeat domain containing 7 (fbxw7) gene. Importantly, stl64 mutants phenocopy previously described fbxw7vu56 mutants, and knockdown of fbxw7 in wild-type animals produced similar defects, demonstrating that stl64 disrupts fbxw7. Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis