Atoh7 cis regulation during retinal ganglion cell development

University Of California At Davis, Davis CA

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Abstract

Project Summary/Abstract Retinal ganglion cells (RGCs) connect the eyes to the brain and are essential for vertebrate vision. One therapeutic goal of vision scientists is to fully understand the factors required for RGC development, so these cells can be generated in vitro. The proneural basic helix-loop-helix (bHLH) protein ATOH7 is expressed transiently in a subpopulation of early retinal progenitor cells and an essential competence factor for RGC genesis. Loss of ATOH7 causes optic nerve aplasia and severe retinovascular malformations. In humans with nonsyndromic congenital retinal nonattachment (NCRNA), a remote 5' conserved enhancer for ATOH7 is deleted, preventing development of RGCs, leading to total blindness from birth. This DNA segment is obviously vital, but its exact role is unknown. In preliminary transgene reporter experiments, this `shadow' enhancer appears to be wholly redundant with the `primary' (promoter-adjacent) enhancer. In this project, I will investigate ATOH7 transcriptional regulation in detail, to understand how specific DNA sequences control the level, timing and pattern of expression. As RGCs are the first-born retinal neurons, these dual enhancers effectively determine the onset of neurogenesis in the vertebrate eye. How does the shadow enhancer influence Atoh7 expression at transcriptional and chromatin levels to ensure RGC development occurs? To evaluate shadow enhancer function, I will generate a mouse model of NCRNA disease, using CRISPR/Cas9 methods to delete this segment in fertilized oocytes, and investigate how the deletion affects retinal histogenesis, Atoh7 expression and regional chromatin status during development. Transcription will be assessed by two methods ? [1] an internally controlled competitive RT-PCR, in heterozyotes carrying the SEdel or wild-type allele with an Atoh7HA knock-in allele in trans, and [2] qPCR analysis of Atoh7 mRNA abundance in an allelic series of wild-type, KO and SEdel embryos. Atoh7 chromatin landscape will be evaluated by chromatin conformation capture (3C) and ATAC-seq methods, to profile DNA looping and nucleosome exposure, respectively. These data will extend our knowledge of Atoh7 regulation, the onset of retinal neurogenesis, RGC fate specification, and the action of binary enhancers generally.