Delineation of Semaphorin6a/PlexinA2 Signaling in Zebrafish Eye Development

University Of Vermont & State Agricultural College, Burlington VT

Investigators

Abstract

As an embryo develops its organs, the cells that form the organs increase in number and take on different functions. Intricate developmental processes are in place allowing cells to communicate with each other to specify the number and types of cells required to form a functioning organ of the appropriate size. This project will study two specific proteins that are required to form an eye. One protein is on one cell and the other is on a neighboring cell and the two proteins communicate with each other. The Principal Investigator hypothesizes that these proteins control cell proliferation and cell type specification during eye formation. Zebrafish embryos will be used for these studies because they are transparent and allow examination of eye development in real time. The proteins that govern eye development in the zebrafish are the same proteins that govern eye development in all vertebrates. The project aims to understand how this critical pair of proteins directs the cells to proliferate and become functional. Undergraduate and graduate students will be trained in research. High school seniors from Puerto Rico will be selected to participate in an eight-week research program and to work on projects related to the proposed research. The researchers and their graduate students will create and direct inquiry based teaching modules at four primarily undergraduate institutions. Students will have opportunities to learn about zebrafish development and the effects of different environmental factors on development. The Principal Investigator demonstrated that a cellular signaling cue, Semaphorin6a, and its receptor, PlexinA2, are required for development of the neuronal tissues of the vertebrate eye. Inhibition of either of these proteins results in smaller eyes due to retinal cells abnormally exiting the eye domain and due to decreases in cell division during the early stages of eye formation. Wild type and mutated Plexin A2 receptors will be expressed in cultured cells. The cells will be stimulated with Semaphorin 6a and modifications to the receptors and changes in binding partners will be determined by imunoprecipitation, immunoblotting, and mass spectrometry. To validate the cell culture studies, PlexinA2 receptors will be expressed in both PlexinA2 null and morphant zebrafish. These studies will focus on answering whether 1) Fyn kinase directly binds to PlexinA2 via its SH3 domain, 2) Fyn and Semaphorin6a induce phosphorylation of PlexinA2 at key regulatory sites, and 3) phosphorylation of the receptor at these sites recruits binding partners responsible for propagation of the signal and the transcriptional regulation of genes involved in eye development. A second aim will be to determine how the Semaphorin6a/PlexinA2-dependent gene targets control cohesion and proliferation in the early zebrafish eye.