Characterizing migratory cell differentiation in developing zebrafish skin

About This Grant

Abstract Mature cell types must be properly differentiated and distributed in tissues during development in all organisms, including humans. Some examples of cells that are broadly distributed include mucus cells (also called goblet cells) and ionocytes, which populate many human mucosal tissues, including the lung. Mucus cells produce mucus to lubricate and protect the epithelial surface; ionocytes regulate the ion balance on either side of the epithelial barrier. Dysregulation of either of these cell types causes severe human disease in many organ systems. Analogous cell types are also found in embryonic zebrafish skin, a highly tractable model system in which live imaging can be easily used to study development. This proposal will characterize the development of recently discovered intraepithelial migratory cells in embryonic zebrafish skin that are precursors to mucus cells and ionocytes, dissecting their mechanism of migration as well as the gene expression patterns responsible for their differentiation. The Sagasti lab has found that these cells derive from stem-like tp63+ basal cells in zebrafish skin during the first day of development, migrate between the basal and periderm layers of skin throughout the body for several hours, and eventually halt and intercalate into the periderm, where they become differentiated mucus cells or ionocytes. We propose that this process functions to spatially distribute mature ionocytes and mucus cells throughout the skin and may be conserved in human mucosal tissues. In this proposal, I will use live imaging and antibody staining to determine whether these migratory cells exhibit mechanisms of amoeboid or mesenchymal migration, as well as pharmacologic and genetic inhibitions to functionally characterize these pathways. Additionally, I will use single-cell RNA sequencing and pseudotime analysis to describe the process by which these cells differentiate into mature cell types and generate candidate genes that are involved in migration. Finally, I will use a CRISPR screen to find genes that are required for migratory cell motility and distribution, and create knockout lines for future study. Taken together, this project will investigate a method of simultaneous cell migration and differentiation within a developing epithelial tissue, resulting in the proper distribution of mature cells. My training goal is to be an independent investigator in the field of skin developmental biology. The proposed fellowship training is an important step towards this career. The research proposed here will give me an excellent background in zebrafish research and epithelial development, training me in impactful techniques, including live imaging, genetic manipulation, and bioinformatics. Further, my training plan includes the IRACDA program at UCLA, which will train me in teaching pedagogy and provide experience designing and delivering lectures at CSULA under the mentorship of experienced professors. This experience will prepare me for the multifaceted career of a principal investigator by gaining experience with zebrafish epithelial research, teaching, and mentorship.