Retinal cell specification is largely controlled by the progression of multi-potent retinal progenitor cells (RPCs) through a series of developmental competence states, during which RPCs successively acquire and lose the ability to generate different cell types. Recent studies have shown that RPC competence is controlled cell- autonomously. This has led to the widely held hypothesis that RPC competence is regulated by temporally dynamic changes in transcription factor expression and activity. However, so far, few transcription factors that regulate RPC competence changes have been identified . We hypothesize that comprehensively profiling mRNA expression in individual RPCs during the course of retinal neurogenesis will identify genes that are both diagnostic of different competence states and that control competence transitions. To test this hypothesis, single cell RNA-Seq of a time-series of RPCs will be conducted to address changes in transcript expression that correlate with changes in RPC competence, across retinal development, at the level of individual cells. Bioinformatic clustering analyses will be used to uncover heterogeneity of RPCs and transcript identifiers of clustered RPCs. Transcripts that differentiate individual clusters of RPCs will be characterized in further experiments. Validation of expression of candidate genes will be examined through in situ hybridization, qRT- PCR and lineage restriction studies. Regulation of RPC competence by candidate genes will be addressed through gain and loss of function analyses using in vivo electroporations. Identification of protein binding partners that participate in the regulation of RPC competence will be performed using protein arrays. Finally, ChiRP-Seq, ChIP-Seq, and MPRA experiments will be conducted to address mechanisms directing RPC competence by candidate genes. These studies will provide important insight into genes and mechanisms regulating neural progenitor competence. Importantly, these studies aim to determine the mechanisms controlling cell type specification, information vital to the generation of pure populations of transplantable cells for treatment of retinal dystrophies.
Identification of molecular mechanisms mediating intrinsic control of retinal progenitor competence Applicant: Brian S Clark, PhD Public Health Relevance Statement (Project Narrative): 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.
| de Melo, Jimmy; Clark, Brian S; Venkataraman, Anand et al. (2018) Ldb1- and Rnf12-dependent regulation of Lhx2 controls the relative balance between neurogenesis and gliogenesis in the retina. Development 145: |
