Purchase this article with an account.
Ann C. Morris, Marie A. Forbes-Osborne; Investigating The Role Of Insm1a In Photoreceptor Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5424.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Insm1 is a conserved zinc finger transcriptional repressor expressed throughout the developing nervous system. Insm1 is expressed in the developing vertebrate retina, however its function is unknown. Insm1 can regulate bHLH transcription factors, suggesting that it is a member of transcriptional regulatory networks that control cell fate determination. Zebrafish possess two co-orthologues of insm1, insm1a and insm1b. Here, we examined the expression and function of insm1a during retinal development.
Gene expression was studied by whole mount in situ hybridization. Antisense morpholinos were injected into zebrafish embryos to knock down expression of insm1a. Morphant embryos were analyzed by light microscopy, in situ hybridization, and immunohistochemistry to determine the effects of insm1a knockdown on retinal and photoreceptor development.
Expression of insm1a was first detectable in the retina between 24 and 28 hours post fertilization (hpf). At 36 hpf insm1a expression was observed throughout the central retinal neuroepithelium. Expression of insm1a shifted to the peripheral retina at 48 and 72 hpf, with stronger expression in the dorsal compared to the ventral retina. We did not observe expression of insm1a in mature photoreceptors. Double in situ hybridization with probes for insm1a and PCNA at 48 hpf revealed that the population of insm1a+ cells was located adjacent to the PCNA+ cells at the retinal margin with minimal overlap between the two, suggesting that insm1a is expressed mostly in postmitotic cells. Transgenic XOPS-GFP embryos injected with a translation-blocking insm1a morpholino displayed no overt morphological defects at 48 hpf. However, we did measure a small but significant decrease in the thickness of the developing retina when compared to control embryos. At 3 days post fertilization (dpf) we observed significantly less GFP fluorescence in the eyes of insm1a morphants compared with controls, indicating fewer numbers of GFP+ rods. This was confirmed by performing immunolabeling with cell-type specific markers in sections of morphant and control retinas at 76 hpf. We did not observe a difference, at least qualitatively, in the number of cells that immunolabeled for the cone marker Zpr-1 or the ganglion cell/amacrine cell marker HuC/D.
Our data show that knockdown of insm1a results in a rod photoreceptor-specific deficiency in the developing retina. Taken together, our results suggest that insm1a expression is required for proper differentiation of rod photoreceptors, and that insm1a acts during the transition of mitotic retinal progenitor cells into post-mitotic photoreceptor precursors.
This PDF is available to Subscribers Only