Mutation of differentiation genes Prkci or Foxo3 modifies the retinal dysplastic phenotype of Crb1^rd8 mice

Abstract

Purpose : Variants of apicobasal polarity determinant CRB1 are associated with retinal dysplasia in humans and in mice. In both species, evidence suggests that genetic modifiers influence the severity of dysplastic disease. The purpose here is to identify genetic modifiers in mice that increase retinal dysplasia in the presence of the homozygous Crb1^rd8 allele.

Methods : Candidate genetic modifiers of the Crb1^rd8 phenotype were identified by indirect ophthalmoscopy of strains generated by chemical mutagenesis of inbred B6.Cg-Crb1^rd8 mice, or of strains developed by the Knockout Mouse Phenotyping Project (KOMP²) at The Jackson Laboratory in C57BL/6NJ mice, which are naturally homozygous for the Crb1^rd8 allele. The fundus phenotype was validated by brightfield fundus imaging, optical coherence tomography (OCT) and histological analysis of retinal sections. Epistasis testing was performed by intercrossing modifier strains with C57BL/6J or C57BL/6NJ-Crb1^rd8+em1Mvw/MvwJ mice, in which the rd8 mutation was corrected. The causal modifier on the B6.Cg-Crb1^rd8 background was identified by mapping, high-throughput sequencing and mutation analysis.

Results : A B6.Cg-Crb1^rd8 mutant strain, Tvrm323, showed an increased frequency of Crb1^rd8-like retinal lesions. The mutation was semidominant and mapped to Chr 3. Whole-exome sequencing revealed a c.406T>A transversion mutation in the Prkci gene, corresponding to p.Tyr136Asn. Epistasis testing showed that the Prkci^Tvrm323 retinal phenotype requires homozygosity at the Crb1^rd8 locus. OCT and histology indicated that the lesions consist of dysplastic foci characterized by thickening of the outer nuclear layer and distortion of inner retinal layers. A KOMP² strain carrying a homozygous Foxo3^{tm1.1(KOMP)Vlcg} knockout allele showed an increased incidence of Crb1^rd8-like lesions by indirect ophthalmoscopy and fundus imaging. Epistasis testing indicated that this phenotype required the homozygous Crb1^rd8 allele, and OCT and histology validated the dysplastic phenotype.

Conclusions : Mutations in Prcki or Foxo3 increase the severity of retinal dysplasia in association with the homozygous Crb1^rd8 allele. The shared role of these genes in apicobasal polarity and in balancing progenitor cell renewal against asymmetric division is intriguing, and may provide clues to cellular differentiation processes that contribute to CRB1/Crb1-dependent retinal dysplasia.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.