*** Morrison IBN 95-13655 This is a proposal to study the development of pigment pattern in the zebrafish, Danio rerio. The zebrafish skin is an excellent model system for studying pattern formation. Initially, an embryonic pattern of four black stripes emerges during early development and over a period of several weeks the pattern is progressively transformed into the alternating blue-black and silvery-yellow striped pattern of the adult fish. The pigment pattern is composed of multiple identifiable types of pigmented cells (chromatophores) that are arranged very specifically with relation to one another such that particular tissue colors are expressed in particular areas of the skin. This study will examine the changes in morphology and migration of the chromatophores identified in normal fish and in a variety of mutant pigment phenotypes. The PI will also address the spatial interdependence of chromatophores required to produce specific pattern elements. This will require a comprehensive analysis of the movements and relative position of the various chromatophore types (both vertically and horizontally in the skin). The studies will also address which chromatophore types or morphologies are derived from common cellular progenitors. Chromatophores combine their spectral properties to produce specific tissue colors. The arrangement, movements and the ultrastructure of these cells will be assessed using several different methodologies. Light microscopy and spectrophotometry will be used to observe and quantify tissue colors. Transmission electron microscopy (TEM) will be used to qualitatively and quantitatively determine the distinctive ultrastructure of chromatophores. High resolution time lapse video will be employed to follow the trajectories of the chromatophores during pattern formation. In addition, high performance liquid chromatography techniques will be used to separate, identify, and quantify pigments extracted from tissues. Together these techniques will provide a thorough multilevel determination of the cellular and biochemical basis of a given pattern and the associated tissue color. A comprehensive morphological framework with which to interpret pattern alterations is not available, despite the fact that several laboratories around the world are generating pigmentation mutants. The goal of this application is to provide the fundamental morphological data required to analyze mutant pigment patterns. This research program is a part of a balanced approach to an academic career and is also an integral part of an educational program as well. The project will enhance opportunities for undergraduates at Hood College, a single-gender woman's institution, to participate in an active research program. The research plan is designed so that undergraduate students gain valuable experience by doing collaborative discovery-based learning. The very nature of determining the precise stage of embryos, preparing the specimens for light optical analysis, the acquisition of digital images and their manipulation using computer software will insure that much of the science effort will be performed by students. This proposal will also allow zebrafish preparations to be included in cell biology and developmental biology classes at Hood College. Funding of this proposal will provide opportunities for women to excel in science. It is also noteworthy that 25% of the enrollment at Hood College are members of under represented minorities. ***
