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Katie Leigh Bales, Wesley R Lewis, Bradley K Yoder, Robert A Kesterson, Alecia K Gross; Retinal degeneration associated with transition zone protein MKS6. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3175.
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© ARVO (1962-2015); The Authors (2016-present)
The transition zone (TZ) within retinal primary cilia plays a major role in proper localization and trafficking of proteins. Mutations occurring in TZ components result in retinal degeneration-associated ciliopathies. The exact role of the TZ gene Meckel Grüber 6 (MKS6 or CC2D2A) is currently unknown within the retina.
To generate a conditional Mks6 allele, embryonic stem cells containing LoxP sites flanking exons 6 and 7 of the Mks6 allele were obtained from MMRC. Mice positive for the Mks6F allele were then mated to homozygosity (Mks6F/F) and crossed with CAGG-Cre; Mks6F/+ mice to generate the Mks6 CAGG conditional mutant line. To induce Mks6 loss, mice were injected once at a dose of 6mg/40g tamoxifen at p7 for juvenille induction. Adult induced mice were injected with 5 daily doses of 6mg/40gtamoxifen starting at p56. Mice were genotyped to confirm deletion and euthanized for analysis. To test for protein localization, eyes were enucleated, fixed, sectioned, stained and imaged using confocal microscopy. For dark adaptation studies, mice were dark adapted for 24 hours prior to euthanasia and analysis.
In dark-adapted conditions, p21 MKS6 conditional knockout (KO) mice experience slight mislocalization of arrestin; found in both the inner and outer segments of photoreceptors whereas rhodopsin and transducin are properly localized in the dark. Under lighted conditions all proteins monitored localized normally. Retinas from these animals displayed abnormal outer segment (OS) length. Staining with wheat germ agglutinin showed that the conditional MKS6 mutant mice had statistically significant shortened OS, although there were no differences in the number of nuclei present in the outer nuclear layer (ONL). In contrast, MKS6 conditional KO mice at 6 months have only one row of nuclei in the ONL, with minimal OS present. Mislocalization of arrestin, transducin and rhodopsin occurs in these older mice.
Based on these results, MKS6 plays an important role in not only proper trafficking of retinal proteins but overall retinal homeostasis. In juvenile induced conditional MKS6 mice, under dark conditions, arrestin is found in both the inner and outer segments. Statistical significant differences were found on OS length between wild type and conditional KO mice at p21 and 6 months. These data support the hypothesis of the importance of MKS6 within the TZ in maintenance of the primary cilia in the retina.
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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