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Stephen Wilson, Marie Forbes-Osborne, Ann Morris; Transcriptional regulation by Her proteins during vertebrate retinal development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3754. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Hairy/Enhancer of Split Related (her) transcription factors are expressed during embryonic neurogenesis and regeneration of the zebrafish retina. Many her genes are direct targets of Notch-Delta signaling, and act as effectors of the Notch pathway to repress transcription of pro-neural genes. Here we investigated the expression of her4, her9, and her12 during retinal development and regeneration, and characterized these genes in the context of genetic pathways.
The expression patterns of three her genes during embryonic retinal development and in the adult retina were characterized using in situ hybridization (ISH). Pharmacological inhibition and genetic overexpression of Notch signaling were accomplished using DAPT and the hsp70-Gal4/UAS-NICD system, respectively. Dual-luciferase assays were performed with her expression vectors and target promoter-luciferase reporters to identify direct targets of her transcriptional regulation. Translation blocking morpholinos were injected into 1-cell stage embryos to knock down expression of specific her genes.
Her4, 9, and 12 demonstrated overlapping yet distinct expression patterns in the developing retina. Expression of her9 was also observed in the retinal vasculature. Expression of her4, 9, and 12 was restricted to the proliferative ciliary marginal zone in the differentiated retina. Manipulation of the Notch-Delta pathway resulted in expression changes in her4 and her12 but not her9. Expression of the neural differentiation genes insm1a and sox11b increased when Notch-Delta signaling was inhibited. Luciferase assays showed that her4 and her9 can directly repress insm1a and neuroD in vitro. Knock-down of her9 resulted in a loss of retinal vasculature.
Our results show that expression of her4, her9, and her12 correspond with proliferative zones of the retina. Her4 and her12, but not her9 are Notch-responsive. In vitro assays showed that her genes repress expression of genes that promote differentiation, consistent with the hypothesis that her genes repress neurogenesis and are required for stem cell maintenance in the retina. In addition, her9 is expressed in retinal vasculature during development, and knock-down of her9 results in reduced retinal vasculature, suggesting that it plays a role in promoting vasculogenesis.
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