Identification of the molecular mechanisms mediating intrinsic control of retinal progenitor competence

NIH RePORTER · NIH · R00 · $248,900 · view on reporter.nih.gov ↗

Abstract

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.

Key facts

NIH application ID
10011818
Project number
5R00EY027844-04
Recipient
WASHINGTON UNIVERSITY
Principal Investigator
Brian S Clark
Activity code
R00
Funding institute
NIH
Fiscal year
2020
Award amount
$248,900
Award type
5
Project period
2017-04-01 → 2022-05-31