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D.C. Otteson, S. Gray, M. Jain, T. Wensel, J. Wilson, D.J. Zack; Mice Lacking the Transcriptional Repressor KLF15 Show a Limited, but Statistically Significant Increase in Ectopic Rhodopsin Expressing Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3970.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The zinc–finger transcription factor KLF15 functions in vitro as a repressor of the Rhodospin promoter and is co–expressed with CRX in the inner nuclear layer. The present studies were designed to test the hypothesis that KLF15 represses Rhodopsin expression in non–photoreceptor cells in vivo by analyzing Rhodopsin–GFP expression in KLF15–lacZ/RhoGFP double mutants. Methods: F1 progeny from matings of homozygous KLF15–lacZ mice (in which exon 2 of KLF15 was replaced by ß–galactosidase) and homozygous RhoGFP mice (in which the endogenous Rhodopsin gene was replaced by a human Rhodopsin–GFP fusion construct) were pair–mated and genotyped. Pups from matings of recombinant and non–recombinant F2 sibs were analyzed at P28. Unfixed cryosections, cut at 10 µm, were photographed under epifluorescence illumination. RhoGFP positive cells in the INL were counted in 1 central and 1 peripheral field in 8 non–adjacent, unfixed retinal sections that were masked for genotype and compared using Mann–Whitney U test. Results: LacZ expression in the retina of heterozygous KLF15–lacZ mice was initally detected in inner nuclear layer cells at P5 and staining intensity increased to approximately adult levels by P28. Although only 50% of expected homozygotes were recovered from matings of KLF15–lacZ mice in a C57/Bl6 background, when outcrossed to RhoGFP, all genotypes were recovered at expected Mendelian ratios. At P28, RhoGFP expression in the outer nuclear layer of mice homozygous, heterozygous or wild–type for KLF15–lacZ was indistinguishable. All eyes contained ectopic RhoGFP expressing cells in the inner nuclear layer; these appeared as either isolated cells or small clusters with diffuse RhoGFP expression throughout the cell membrane and a single, polarized accumulation of strong RhoGFP. On average, there were 2.5 RhoGFP–positive cells per 640 µm retinal length in homozygous KLF15–lacZ mice compared to 1.3 for their wild–type sibs (p=0.029). Conclusions: There is a small, but statistically significant, increase in the number of RhoGFP expressing cells in the inner nuclear layer of KLF15–null mice at P28. However, in the majority of non–photoreceptor cells, RhoGFP was not detected. These results are consistent with a role for KLF15 in repressing Rhodopsin expression in the INL, but the magnitude of the effect suggests that additional factors may be involved.
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