Mechanisms of photoreceptor specification and morphogenesis

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PROJECT SUMMARY Despite our dependency upon cone-mediated vision, and the debilitating impact of cone degeneration upon our central vision, major gaps exist in our knowledge of genes and mechanisms that regulate the number and spatial patterning of photoreceptors. Underlying this asymmetry is the fundamental choice of retinal neuroblasts to re-enter mitosis or differentiate. The larval zebrafish retina provides an unparalleled genetic model to identify fundamental mechanisms integrating photoreceptor specification and spatial patterning. The larval zebrafish retina is anatomically and functionally cone-dominated with 4 cone subtypes arranged in a highly ordered mosaic. Rods are far less numerous and distributed asymmetrically along the dorsal/ventral axis. The range of genetic tools, availability of genomic resources and access to the zebrafish embryo, permit a systematic approach to uncover the impact of genetic manipulations upon cell fate and their distribution. Our premise is based upon rigorous genetic and molecular analysis in our published studies and preliminary data showing that tbx2b/lor and six7/ljr are essential for maintaining the cone-dominated zebrafish retina. Mutations of tbx2b result in a 5-fold increase and uniform distribution of rods due to a cell fate switch of SWS1- cones into rods, a phenotype opposite to that of the rd7 mouse and enhanced-S cone syndrome in humans. Mutations of six7, an orthologue of Six3/6 result in a similar increase and uniform distribution of rods, however characterization of several alleles revealed that six7 independently suppresses mitosis of late stage progenitors, and is essential for survival of a cone subtype. Our goal is to understand the mechanisms underlying functions of tbx2b and six7 in cell fate decisions and spatial patterning in the context of know factors that regulate photoreceptor development. Aim 1 will take advantage of innovative genome editing tools, transposon-based transgenesis, and more routine methods to test the hypothesis that six7 modulates the choice of late stage photoreceptor progenitors between continued mitosis or differentiation. Aim 2 combined in vivo and in vitro approaches to test our novel hypothesis that tbx2b regulates the timing of photoreceptor determination and thereby spatial patterning through molecular interaction with known photoreceptor transcription factors. Furthermore, our unpublished data and published reports show unexpected alterations in gene expression following genetic alterations suggesting unrecognized roles for these factors in maintaining cell fate. Therefore, completion of the specific aims will significantly advance the fields knowledge of mechanisms regulating the generation of a highly specialized, cone-dominated retina.