Vertebrate Photoreceptor Development and Regeneration

Retinitis pigmentosa (RP) and allied retinal dystrophies comprise a heterogeneous group of genetic disorders resulting in photoreceptor degeneration and blindness, for which there is currently no cure. Many RP-causing mutations are inherited in an autosomal dominant manner, rendering gene therapy approaches a challenge. Furthermore, gene therapy will only work at the early stages of the disease when a significant number of surviving photoreceptors remain. One promising alternative therapeutic approach is cell-based transplantation therapy, whereby photoreceptor precursor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of photoreceptor precursors into the recipient retina, and the difficulty of obtaining sufficient numbers of such cells for clinical application. Protocols for the in vitro culture of retinal progenitor cells (RPCs) must be developed that will produce large numbers of photoreceptor precursors and will promote their survival and differentiation once transplanted. Recent studies have shown that modulation of transcription factor (TF) expression in RPCs can influence their adoption of a photoreceptor fate and their integration upon transplantation. Therefore, defining the transcriptional networks that promote specification and differentiation of photoreceptor precursors is a necessary next step for the evolution of cell transplantation therapies. A long-term goal of our laboratory is to define at a molecular level the gene regulatory networks that control photoreceptor subtype development and regeneration. We use the powerful zebrafish model, which allows the application of molecular genetic approaches to a vertebrate system with the capacity to regenerate retinal neurons. Using a transgenic line of zebrafish that displays specific rod photoreceptor degeneration and regeneration, we previously identified several transcription factors (TF’s) that are induced during rod regeneration, whose function during photoreceptor development was previously unknown. For the past several years, we have been systematically studying these TF’s using a combination of molecular, genetic, biochemical, and cellular imaging methods. In this proposal, we will continue this successful approach by focusing on three TF’s that we hypothesize are required for proper rod photoreceptor differentiation: Sox4, Sox11, and Her9. Specific Aim 1: Determine the role of the SoxC transcription factors Sox4 and Sox11 in rod photoreceptor differentiation. Previously, we demonstrated that Sox4 and Sox11 regulate ocular morphogenesis and rod photoreceptor development through inhibition of Hh signaling. To investigate the long-term consequences of loss of SoxC activity, we have been generating targeted mutations in each SoxC co-orthologue (sox4a, sox4b, sox11a, and sox11b) using CRISPR/Cas genome editing. Our preliminary data reveal that loss of sox4a results in altered Hh and Bmp signaling in the early embryo, which is accompanied by microphthalmia and a sustained reduction in rod photoreceptor number. Intriguingly, our data suggest that Sox4 is required prior to the optic cup stage for proper rod photoreceptor differentiation. In this aim, we will 1) determine whether there is a dosage effect of SoxC activity on rod photoreceptor number; 2) use state-of-the-art time-lapse imaging to determine when and where SoxC expression is required to achieve proper rod photoreceptor development; 3) determine precisely how SoxC factors regulate Hh and Bmp signaling; 4) identify the molecular targets of SoxC factors; and 5) determine whether loss of Sox4 impairs adult rod photoreceptor differentiation. Specific Aim 2: Specific Aim 2: Determine the extent and cause of photoreceptor defects in the zebrafish her9 mutant. Her9 is a bHLH-O transcriptional repressor and is the zebrafish homolog of human Hes4. As Hes4 is absent from the mouse genome, zebrafish provide a unique opportunity to explore the function of this TF during retinal development. Intriguingly, although Her9 is a member of the Hes/Hey/Her family of TF’s, which are classic effectors of the Notch pathway, Her9 is not responsive to Notch signaling. We generated her9 mutants, and found that loss of her9 results in absence of the larval visual background adaptation response (suggestive of a defective visual system) as well as reduced rod photoreceptors and altered cone photoreceptor morphology. In this aim, we will 1) determine the mechanism for photoreceptor defects in her9 mutants; 2) determine whether loss of Her9 impairs vision; 3) identify the upstream signaling pathway(s) that regulate her9 expression in the retina; and 4) determine whether Her9-mediated VEGF expression in the avascular zebrafish retina regulates retinal progenitor cell proliferation and differentiation. We predict that the successful completion of these aims will reveal some of the earliest events that bias RPCs towards the photoreceptor lineage, as well as uncover novel genetic pathways and signaling mechanisms that promote photoreceptor fate.