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Marie Forbes-Osborne, Stephen Wilson, Ann Morris; The transcription factor Insm1 regulates photoreceptor differentiation and cell cycle progression. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3752.
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Insulinoma-associated 1 (Insm1) is an evolutionarily conserved zinc-finger transcription factor. Insm1 is expressed throughout the developing nervous and neuroendocrine systems, including in the developing retina. Additionally, insm1 is upregulated in response to chronic rod photoreceptor degeneration and in cancers. While Insm1 has known functions in pancreatic beta cell development, its role in retinal development has not been determined. Similarly, few direct targets of Insm1 have been identified, and little is known about the mechanism of Insm1 transcriptional regulation. In this study, we examined the function of insm1a during zebrafish retinal development.
Antisense morpholinos were injected into zebrafish embryos to knock down expression of insm1a. Morphant embryos were analyzed by light microscopy, immunohistochemistry, and in situ hybridization to determine the effects of insm1a knockdown on retinal neurogenesis. Cell cycle kinetics in insm1a morphants were examined using immunohistochemistry for markers of S and M phase. In vitro luciferase reporter assays were performed to determine whether Insm1a could directly regulate expression of its potential target genes in vitro.
Knockdown of insm1a caused a significant and specific decrease in eye area at 48 hpf. Further analysis showed a significant decrease in differentiated rod and cone photoreceptor cells at 3 days post fertilization (dpf), with rod photoreceptors remaining significantly reduced at 4 dpf. Muller glia and retinal ganglion cell numbers were unchanged at 3 dpf, but other retinal cell types showed slight reductions in number, and immature morphology. Cell cycle changes were also observed with insm1a knockdown. Changes in expression of known effectors of retinal development (including ath5/atoh7 and neurod) were observed, and Insm1a directly regulated their expression in vitro.
Our data show that knockdown of insm1a results in a rod photoreceptor deficiency in the developing retina, accompanied by reduced neuronal maturity, and changes in cell cycle kinetics. Taken together, these results suggest that insm1a is required for rod photoreceptor differentiation, and that insm1a expression is necessary for proper cell cycle progression.
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