Even though proper vision is important to all types of animals, how the parts of the eyes that communicate with the brain (the retinas) develop is only partly understood. The present research project is conducted in zebrafish because of advantageous genetic and experimental tools available for studying early eye development. Previous studies carried out by this research team found two proteins that need to function properly for retinas to develop (called DCBLD1 and DCBLD2); if these proteins aren't made, retinas don't form properly. Replacing a defective fish DBCLD2 protein with a human DCBLD2 protein enables the fish retina to develop normally, suggesting that DCBLD proteins function in a similar manner in all animals with backbones. The present project will examine the mechanisms by which DCBLD proteins govern proper retina formation using a combination of anatomical, biochemical, cell biological, functional genetic and proteomic techniques, and will provide a more complete understanding of retina formation. Broader impacts of this research include laboratory training for high school students, university undergraduates, graduate students, and staff scientists in many modern experimental approaches. Several of the students will be specifically recruited from populations that are underrepresented in science. The research and training will be conducted by an inter-disciplinary team in Vermont (the University of Vermont and Norwich University), Massachusetts (Boston College) and Puerto Rico (the University of Puerto Rico-Rio Piedras and the Metropolitan University).
Previous work by the principal investigators determined that the putative neuronal guidance receptors DCBLD1 and 2 act as scaffolds for the Crk family of adaptors, which are known to play important roles in the positioning of neurons during development. Histological studies in transgenic zebrafish identified Dcbld1 and 2 as essential for both proper retinal ganglion cell development and proper positioning and/or differentiation of retinal cell types. Human DCBLD2 was shown to rescue dcbld2 morphant phenotypes. The following research aims will be conducted to delineate the roles of Dcbld1 and 2 in zebrafish eye development: Aim1 will use immunohistochemistry to determine how loss of Dcbld1 and 2 affects cell proliferation and the differentiation of zebrafish retinal cell types. Aim 2 will use functional developmental approaches to determine if the DCBLD1 and 2 tyrosine phosphorylation sites required for Crk/CrkL binding are essential for the development of the zebrafish retina. Aim 3 will use biochemical, proteomic, cell biological and functional genetic approaches to characterize the phosphorylation of DCBLD proteins, DCBLD binding partners and the roles these play in zebrafish neural retina development.
