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.

Public Health Relevance

I will investigate the structure and function of the ATOH7 gene and its shadow enhancer, a remote DNA segment that controls ATOH7 expression during retinal development, to better understand the pathogenic mechanism for human nonsyndromic retinal nonattachment (NCRNA) disease. The project, which uses mutant laboratory mice, will also advance the NEI goal to improve the efficiency of generating retinal ganglion cells (RGCs) in vitro. By understanding the regulatory network influencing action of the RGC competence factor ATOH7, I will provide other researchers with important new information to achieve this objective.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32EY028003-02
Application #
9664518
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Agarwal, Neeraj
Project Start
2018-01-01
Project End
2019-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618