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Published on: December 22, 2014
A framework to identify functional interactors that contribute to disrupted early retinal development in Vsx2 ocular
Amanda M Leung1, Mahesh B Rao2, Nathan Raju2
1Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA.
Background:
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.
Results:
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.
Conclusions:
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.
Insights
Investigating Vsx2 gene interactions in retinal development, this study found Mitf partially explains the ocular retardation J (orJ) phenotype. Gamma-secretase inhibition revealed convergent negative epistasis with Vsx2, impacting tissue growth.
Area of Science:
- Developmental genetics
- Molecular biology
- Transcriptomics
Background:
- Understanding gene interactions is crucial for explaining mutation-induced phenotypes in developmental genetics.
- The study focuses on Vsx2, a gene critical for early retinal development, using the ocular retardation J (orJ) mouse model.
- Epistasis principles were applied to bulk transcriptome data to identify functional interactors of Vsx2.
Purpose of the Study:
- To identify functional interactors of Vsx2 that influence retinal development.
- To investigate the roles of Mitf, Retinoid-X Receptors (RXR), and gamma-secretase in relation to Vsx2 function.
- To elucidate the mechanisms of positive and negative epistasis in the context of Vsx2 mutations.
Main Methods:
- Utilized the Vsx2 loss-of-function mouse model (orJ) for in vivo studies.
- Performed transcriptome analysis on retinal tissues.
- Applied inhibitors of Retinoid-X Receptors (RXR) and gamma-secretase to ex vivo retinal cultures.
Main Results:
- Mitf demonstrated robust positive epistasis with Vsx2 but only partially explained the orJ phenotype.
- RXR inhibition showed minimal evidence of epistasis between Vsx2 and Rxrg.
- Gamma-secretase inhibition revealed significant Vsx2-dependent gene alterations related to proliferation, indicating convergent negative epistasis.
Conclusions:
- Functional interactions between Vsx2, Mitf, RXR, and gamma-secretase activities were characterized quantitatively and qualitatively.
- The findings highlight the complex genetic network regulating retinal development.
- Transcriptome analysis combined with in vivo and ex vivo experiments provides a powerful approach to dissecting gene function.
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