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Joel B Miesfeld, Li-En Jao, SUMAN Kalyan MANNA, Robert J Zawadzki, Nicholas Marsh-Armstrong, Nadean L Brown, Tom Glaser; Atoh7 cis regulation during retinal ganglion cell development: A multi species approach. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6408.
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© ARVO (1962-2015); The Authors (2016-present)
Nonsyndromic Retinal Nonattachment (NCRNA) is caused by a 6.5 kb deletion located 15-21 kb upstream of the retinal ganglion cell (RGC) competence factor gene ATOH7. NCRNA patients have no RGCs or optic nerves. The deletion spans three conserved noncoding elements (CNE), which together recapitulate the transient expression pattern of endogenous Atoh7, in transgenic mice, and act as a shadow enhancer (SE) (Ghiasvand et al. Nat Neurosci 2011). We generated a murine NCRNA model to test how the SE influences Atoh7 transcription.
We deleted CNE1-3 in C57BL/6J mice by CRISPR/Cas9 editing. To compare Atoh7 transcription from SEdel and WT alleles, we performed competitive triplex RT-PCR assays, using an HA knockin allele in trans, during the onset (E11.5), peak (E14.5) and decline (E16.5, P0.5) of Atoh7 expression in the developing retina. We counted Atoh7+ and Isl1/2+ cells in an allelic series containing Atoh7 WT, SEdel and knockout (KO) retinas at E14.5 by immunofluorescence. Phenotypes were further characterized by H&E histology and OCT imaging. Zebrafish and Xenopus BAC transgenes containing human ATOH7 primary and shadow enhancer segments were evaluated in parallel. Atoh7 chromatin structural effects will be demonstrated by ATACseq and 4C-seq.
The 5.5 kb Atoh7 SE deletion was verified by PCR and Sanger sequencing. We found a uniform 5-fold decrease in Atoh7 mRNA abundance at all timepoints from E11.5 to P0.5. The number of Atoh7+ and Isl1/2+ cells and thickness of nascent optic nerves were significantly decreased in SE/SE and SE/KO eyes at E14.5. Adult SE/KO mice had grossly thin optic nerves and retinal vascular defects. Our analysis of human ATOH7 cis regulatory elements in zebrafish and Xenopus confirms their functional conservation and suggests that CNE1 is dispensable for SE action.
The murine shadow enhancer controls the level of Atoh7 transcription during retinogenesis, but its loss does not cause optic nerve aplasia, the characteristic feature of human NCRNA disease. However, SE/SE and SE/KO animals do have fewer RGCs and secondary retinovascular anomalies, and SE loss disrupts Atoh7 chromatin structure. The zebrafish and Xenopus models allow further dissection of cis regulatory elements.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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