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Amanda G Kautzman, Patrick William Keeley, Caroline R Ackley, Benjamin E Reese; Xkr8 Modulates Bipolar Cell Number in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1779.
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© ARVO (1962-2015); The Authors (2016-present)
The specification of neuronal number in the retina is under precise genomic control, but the underlying genetic mechanisms regulating final population size are still relatively unknown. Through a previous forward genetic screen using recombinant inbred mouse strains, we identified a locus on Chr 4 that was correlated to the variation in neuronal number for two populations of bipolar cell, the rod bipolar cell (RBC) and type II cone bipolar cell (T2CBC). The present study examined a potential role for a candidate gene at this locus, Xkr8, in the modulation of bipolar cell number in the developing mouse retina.
The Chr 4 locus was located at 131.5 Mb, being ~2 Mb wide and containing 55 genes. Each gene was interrogated using bioinformatic tools to assess the presence of genetic variants, retinal expression, and putative gene function. Top candidates were prioritized, and then examined further using in situ hybridization and in vivo electroporation. Adult retinas were probed for the localization of transcripts, while newborn mice were electroporated with control versus overexpression reporter plasmids injected into the subretinal space. At P21, GFP+ cells were counted in different cellular layers across an average of 10 sections per mouse to yield their proportion out of all GFP-expressing cells.
Xkr8 (132.7 Mb) was identified as a candidate gene of interest given the many putative regulatory variants present, expression during retinal development, and a role in the apoptotic pathway. Xkr8 expression levels correlated with RBC and T2CBC number, and variation in expression levels mapped a cis-eQTL. In situ hybridization revealed heavy labeling throughout the ONL and INL, but no labeling in the GCL. Modulation of Xkr8 via overexpression caused a significant decrease in the proportion of GFP+ bipolar cells in the INL (Xkr8: 0.04 + 0.01; Control: 0.09 + 0.01, p = 0.0005), while not altering the proportion of GFP+ amacrine cells (Xkr8: 0.03 + 0.01; Control: 0.04 + 0.01, p = 0.18; n = 9 and 12 mice, respectively).
These results implicate Xkr8 in the establishment of bipolar cell number in the developing retina. Follow-up studies will seek to identify the mechanisms by which Xkr8 controls bipolar cell population size, and to identify the variants responsible for altering gene expression and how they function.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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