June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Characterization of zebrafish her9 regulation and function in retinal development
Author Affiliations & Notes
  • Ann C Morris
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Cagney E Coomer
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Stephen G Wilson
    Biology, University of Kentucky, Lexington, Kentucky, United States
    Biology, University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Ann Morris, None; Cagney Coomer, None; Stephen Wilson, None
  • Footnotes
    Support  NIH Grant EY021769
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 113. doi:
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      Ann C Morris, Cagney E Coomer, Stephen G Wilson; Characterization of zebrafish her9 regulation and function in retinal development. Invest. Ophthalmol. Vis. Sci. 2017;58(8):113.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Her9 is a bHLH-O transcriptional repressor and is the zebrafish homolog of mammalian Hes1. Previously, we found that Her9 expression is upregulated in a background of chronic rod photoreceptor specific degeneration and regeneration in the adult zebrafish retina. In this study, we investigated the function of Her9 in retinal development, identified signaling pathways upstream of her9 expression, and characterized the phenotypes of her9 homozygous mutants.

Methods : All animal procedures were performed in accordance with guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The XOPS:mCFP and fli1:EGFP transgenic lines have been previously described. Wild type or fli1:EGFP embryos were treated with 1 µM retinoic acid (RA), 100 µM of the RA inhibitor DEAB, or 0.3% DMSO alone until 36 hpf and then processed for in situ hybridization (ISH), immunohistochemistry (IHC) or qPCR, which were all performed as previously described. Single strand guide RNA (sgRNA) targeting the her9 locus and Cas9 mRNA were microinjected into zebrafish embryos at the single-cell stage. High resolution melt curve (HRMA) and RFLP analyses were performed to screen for and genotype mutations in her9. Retinal cell type differentiation in wild type and her9 mutants was analyzed by IHC and using fluorescent reporter transgenic lines. Gene expression in control and mutant zebrafish embryos was analyzed by qPCR and by ISH. The visual background adaptation (VBA) assay was performed as previously described. VBA results were analyzed by chi-square analysis.

Results : We found that her9 expression in the developing retina was induced by RA, but not by Notch-Delta signaling. Expression of her9 co-localized with retinal blood vessels, and overexpression of her9 resulted in ectopic retinal vasculature. We recovered a her9 mutant allele that results in a frame-shift and premature termination codon upstream of the bHLH domain. Homozygous her9 mutants diplayed abnormal vasculature, reduced numbers of photoreceptors, a thin or absent retinal ciliary marginal zone, and a poor visual background adaption (VBA) response. Her9 homozygous mutants also failed to develop a swim bladder and did not survive past 10 dpf.

Conclusions : Taken together, our results demonstrate that Her9 is regulated by RA signaling, is required for proper vasculature and retinal development, and loss of Her9 may result in visual impairment.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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