Poster Title

Analysis of hairy-related 9 (her9) during vertebrate ocular development

Grade Level at Time of Presentation

Senior

Institution

University of Kentucky

KY House District #

7

KY Senate District #

7

Department

Dept. of Biology

Abstract

The hairy-related 9 (her9) gene—belonging to the hairy/enhancer of split (hes) superfamily of Basic-Helix-Loop-Helix-Orange (BHLH-O) transcription factors which are involved in many developmental processes—is expressed during vertebrate embryonic retinal development and in the regenerating adult retina. Her9 was found to be upregulated in the retina of a transgenic line of zebrafish that exhibits constitutive rod photoreceptor specific degeneration and regeneration. Her9 has been shown to be expressed throughout the developing central nervous system of the zebrafish, including the retina. Fluorescent in situ hybridization (FISH) experiments were conducted in a transgenic line of zebrafish that expresses the GFP reporter in vascular endothelial cells. We found that her9 expression co-localizes with markers for retinal vasculature. Pharmacological manipulation of several signaling pathways starting during the appearance of the primordial eye field revealed that her9 expression in the retina is sensitive to the Retinoic Acid (RA) signaling pathway. The CRISPR/Cas9 system was used to generate her9 mutant lines. A guide RNA specific to her9 developed in our lab was co-injected with Cas9 mRNA into 1-cell stage zebrafish embryos. These founders were crossed to generate F1 embryos that, upon sequencing, were found to have an insertion or deletion causing a frameshift mutation and no gene product. If our experiments confirm that her9 plays a role in vasculogenesis, this may lead to key therapeutic treatments for eye diseases involving defects in retinal vasculature such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinitis pigmentosa (RP).

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Analysis of hairy-related 9 (her9) during vertebrate ocular development

The hairy-related 9 (her9) gene—belonging to the hairy/enhancer of split (hes) superfamily of Basic-Helix-Loop-Helix-Orange (BHLH-O) transcription factors which are involved in many developmental processes—is expressed during vertebrate embryonic retinal development and in the regenerating adult retina. Her9 was found to be upregulated in the retina of a transgenic line of zebrafish that exhibits constitutive rod photoreceptor specific degeneration and regeneration. Her9 has been shown to be expressed throughout the developing central nervous system of the zebrafish, including the retina. Fluorescent in situ hybridization (FISH) experiments were conducted in a transgenic line of zebrafish that expresses the GFP reporter in vascular endothelial cells. We found that her9 expression co-localizes with markers for retinal vasculature. Pharmacological manipulation of several signaling pathways starting during the appearance of the primordial eye field revealed that her9 expression in the retina is sensitive to the Retinoic Acid (RA) signaling pathway. The CRISPR/Cas9 system was used to generate her9 mutant lines. A guide RNA specific to her9 developed in our lab was co-injected with Cas9 mRNA into 1-cell stage zebrafish embryos. These founders were crossed to generate F1 embryos that, upon sequencing, were found to have an insertion or deletion causing a frameshift mutation and no gene product. If our experiments confirm that her9 plays a role in vasculogenesis, this may lead to key therapeutic treatments for eye diseases involving defects in retinal vasculature such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinitis pigmentosa (RP).