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
