Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

Abstract

The homeodomain and adjacent CVC domain in the visual system homeobox (VSX) proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27 ^Kip1 . Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.

Author Summary

Problems with the early development of the mammalian retina can cause congenital eye defects such as microphthalmia, in which the eye is dramatically smaller and functionally compromised. Severe microphthalmia is associated with mutations in the retinal-expressed visual system homeobox 2 (Vsx2) gene, but how Vsx2 controls retinal development, and ultimately eye formation, has remained unclear. We assessed the impact of two missense mutations, discovered in humans, on Vsx2 function and eye development in mice. One mutation altered a highly conserved residue of the homeodomain, and the other altered a highly conserved residue in the CVC domain, a region of unresolved function. Both mutations impacted the DNA binding properties of the protein, although to differing extents. Likewise, both mutations caused microphthalmia and disruptions in retinal development, also to differing extents and by distinct mechanisms. Our data suggest that Vsx2 acts as a gatekeeper of the retinal gene expression program by preventing the activation of interfering or competing gene expression programs. We propose that the evolutionary stable association between the VSX-class homeodomain and CVC domain set the stage for Vsx2 or its archetype to assume a gatekeeper function for retinal development and ultimately eye organogenesis.