The specification of retinal cell types from a common, multi-potent progenitor cell occurs in an overlapping temporal birth order. Previous studies have concluded that the selection of an individual progenitor to undergo a terminal division, whereby at least one daughter cell exits the cell cycle, is largely controlled cell autonomously. Therefore, it has been hypothesized that changes in the ability of retinal progenitors to differentiate as specific retinal cell types results from either an inherent heterogeneity of progenitors resulting in lineage biases or global changes in gene expression across development that correspond to changes in cell fate specification. In order to test these hypotheses, I previously characterized the transcript expression of individual progenitors across retinal development using single-cell RNA-sequencing. These experiments support a model in which retinal progenitors are not lineage restricted but exhibit global changes in gene expression corresponding to progenitor cell maturation across development. Additionally, the profiling of retinal progenitor transcriptomes has enabled us to identify numerous candidate genes hypothesized to 1) regulate the proliferative potential of retinal progenitors 2) confer maturation of retinal progenitors across developmental time and 3) control the temporal specification of individual retinal cell fates. As proof in principal, we showed that the late progenitor cell enriched NFI transcription factors regulate both proliferative quiescence and generation of late-born cell types. The goal of these continued studies is to identify the mechanisms by which retinal progenitor cells are selected to undergo a terminal division and to determine if these candidate genes also impart biases in specification of individual retinal cell fates. The function of candidate genes in the regulation of cell cycle exit and cell fate specification will be determined through gain/loss-of-function experiments within the developing retina through both in vivo and ex vivo electroporations and genetic models. Mechanistic insights into candidate gene function will be performed through protein arrays to identify interacting proteins, ChIRP-seq/ChIP-seq to examine the RNA-DNA or protein-DNA interactions, respectively, and through reporter assays to identify the temporal activity of cis-regulatory elements. These studies will provide import insights into the genes and mechanisms regulating temporal cell fate specification within the developing retina, information vital to understanding the pathogenesis of retinal dystrophies and for understanding treatment of these diseases.
Over 10% of American adults over 40 suffer from visual impairments of the retina, yet cellular transplant therapies to restore visual function are in their infancy. Significant gaps remain in the procurement of pure populations of transplantable cells. This proposal seeks to identify the molecular mechanisms controlling the differentiation of specific retinal cell types from retinal progenitor cells, thus providing insight pivotal for the design of cellular therapies for retinal diseases.
