Microphthalmia and Disrupted Retinal Development Due to a Knock-in/Knock-Out Allele at the Locus.

PURPOSE

Visual System Homeobox 2 () is a transcription factor expressed in the developing retina that regulates tissue identity, growth, and fate determination. Several mutations in the gene exist in mice, including a spontaneous nonsense mutation and two targeted missense mutations originally identified in humans. Here, we expand the genetic repertoire to include a reporter allele ( ) designed to express beta-Galactosidase (bGal) and simultaneously disrupt function (knock-in/knock-out).

METHODS

We generated a reporter allele with an in-frame fusion to the coding sequence immediately following exon 2. Germline transmission was assessed with genomic DNA PCR and Western blot analysis was used to describe VSX2 expression from the mutant allele (). Eye size quantification and immunohistology were used to describe the embryonic and postnatal retinal phenotypes of homozygous and heterozygous mice. The contribution of to mutant microphthalmia was probed with the semi-dominant negative allele.

RESULTS

The retinal expression pattern of bGal is concordant with VSX2, and the mutant allele is recessive. homozygous mice have congenital bilateral microphthalmia accompanied by defects in retinal development including ectopic expression of non-retinal genes, reduced proliferation, delayed neurogenesis, aberrant tissue morphology, and an absence of bipolar interneurons - all hallmarks of loss-of-function. The allele reduced the severity of microphthalmia caused by the allele. Unexpectedly, the mutant VSX2 protein is stably expressed, and there are subtle differences in eye size and early retinal neurogenesis when compared to the null mutant, .

CONCLUSION

The perdurance of the mutant VSX2 protein combined with subtle deviations from the null phenotype leaves open the possibility that allele is not a complete knock-out. The allele exhibits loss-of-function characteristics and adds to the genetic toolkit for understanding function.