A framework to identify functional interactors that contribute to disrupted early retinal development in Vsx2 ocular retardation J mice.

Abstract

A goal of developmental genetics is to identify functional interactions that underlie phenotypes caused by mutations. We sought to identify functional interactors of Vsx2, which when mutated, disrupts early retinal development. We utilized the Vsx2 loss-of-function mouse, ocular retardation J (orJ), to assess interactions based on principles of positive and negative epistasis as applied to bulk transcriptome data. This was first tested in vivo with Mitf, a target of Vsx2 repression, and then to cultures of orJ retina treated with inhibitors of Retinoid-X Receptors (RXR) to target Rxrg, an up-regulated gene in the orJ retina, and gamma-Secretase, an enzyme required for Notch signaling, a key mediator of retinal proliferation and neurogenesis. Whereas Mitf exhibited robust positive epistasis with Vsx2, it only partially accounts for the orJ phenotype, suggesting other functional interactors. RXR inhibition yielded minimal evidence for epistasis between Vsx2 and Rxrg. In contrast, gamma-Secretase inhibition caused hundreds of Vsx2-dependent genes associated with proliferation to deviate further from wild-type, providing evidence for convergent negative epistasis with Vsx2 in regulating tissue growth. Combining in vivo and ex vivo testing with transcriptome analysis revealed quantitative and qualitative characteristics of functional interaction between Vsx2, Mitf, RXR, and gamma-Secretase activities.