PROJECT SUMMARY/ABSTRACT There are numerous examples of self-assembled patterns in biology, but most research effort has focused on only a few systems (syncytial patterning in Drosophila, adhesion-based sorting of heterogeneous cell populations, gradient-induced specification of homogeneous populations). The goal of this proposal is to identify molecular mechanisms that regulate pigment pattern formation in adult zebrafish. Adult zebrafish pigment cells differentiate from neural crest-derived progenitors, and reciprocal interactions between pigment cells are essential for stripe pattern formation. One class of pigment cells, iridescent iridophores, is responsible for establishing and reiterating the pattern of dark stripes and light interstripes. Two iridophore subtypes occur in zebrafish: “loose,” mesenchymal-like iridophores that associate with melanophores in dark stripes, and “dense” epithelial-like iridophores of light interstripes that drive orange xanthophore differentiation within the interstripes, while also setting the positions of adjacent stripes. The mechanisms that specify loose and dense iridophore subtypes have not been determined. In this proposal, Aim 1 focuses on an innovative hypothesis to account for subtype specification via interactions among iridophores, melanophores and the tissue environment involving two candidate signaling pathways. Aim 2 addresses essential roles for a cell- autonomous factor, identified by forward genetics, in promoting iridophore maturation and, thereby, the essential interactions between iridophores and other pigment cells underlying pattern formation. Approaches will range from classical genetic analysis to time-lapse imaging, and from gene expression analysis in situ to single cell RNA-Sequencing. These studies will provide insights into iridophore differentiation and morphogenesis that will augment our understanding of the genetic basis of cellular behaviors during the dynamic self-assembly of tissue patterns.