We know very little about how differences in developmental mechanisms generate differences in adult form among organisms. Yet such information is vital to understanding patterns of naturally occurring morphological variation, as well as the evolution of form, developmental patterning mechanisms, and gene function. One system that is especially amenable to addressing these issues is the externally visible pigment pattern of fishes in the genus Danio, which includes the developmental genetic model organism, the zebrafish, D. rerio. In these fishes and other vertebrates, pigment cells are derived from the neural crest, a transient population of embryonic precursor cells that also give rise to many other vertebrate cell types and tissues (e.g., the craniofacial skeleton). Because of the many traits to which they contribute, neural crest cells and their derivatives have important roles in human health and disease, and are associated with a variety of malignancies (e.g., melanoma) and congenital abnormalities (e.g., cleft palette). The studies proposed here will use Danio fishes to elucidate developmental, cellular, and genetic mechanisms underlying pigment pattern variation, specifically a pattern of horizontal melanocyte stripes in zebrafish, and an absence of stripes in D. albolineatus. First, a previously identified candidate gene, fms, will be tested for its role in determining whether or not stripes form. These experiments will use molecular analyses of gene sequences and expression, segregation analyses of fms alleles in interspecific hybrids, and mutational and histological approaches to assess interspecific variation in fms-dependent pigment cell lineages. Second, the morphogenetic bases for stripe formation and its evolutionary loss will be determined. These studies will employ image analyses of pigment cell behavior, as well as histological examination of cell death, differentiation, and proliferation; the cellular autonomy of stripe formation and loss will also be determined using interspecific and intraspecific cell transplantation. Finally, additional genes and genetic pathways will be identified that contribute to pigment pattern variation by screening other previously isolated candidate genes, as well as large genomic regions. This will be accomplished by analyzing pigment patterns in hybrids between D. albolineatus and zebrafish homozygous for pigment pattern mutants, as well as previously isolated chromosomal deletions that uncover much of the zebrafish genome. Together, these studies will provide novel insights into the origins of naturally occurring variation in a biomedically significant neural crest derivative, and will be an essential next step in further elucidating the developmental mechanisms of pigment pattern formation in zebrafish and its relatives.
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