Uncovering the genetic determinants of superior coloboma, a novel ocular congenital disorder

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye due to failed choroid fissure closure. Recently, identification of individuals with coloboma in the superior aspect of the iris led to the discovery of a novel developmental structure, referred to as the superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during early vertebrate eye development. Previous research in our lab demonstrated that manipulation of BMP signaling results in SOS closure delay. We aim to elucidate the role of a key dorsoventral (DV) eye axis patterning gene identified in patient #4 exome sequencing data, VAX2, a transcription factor regulated by Shh signaling that is expressed in the ventral eye. Using zebrafish, we created a vax2-null allele, and observe that resultant homozygous mutant embryos display SOS closure delay. Overexpression of wildtype VAX2 mRNA in zebrafish embryos leads to SOS closure delay and perturbed expression of DV-axis patterning genes. Analysis of the patient variant (p.Leu139Met) conclusively demonstrates decreased activity when compared to wildtype VAX2, indicating that the patient variant is likely to be hypomorphic. These findings suggest that SOS closure appears to require a tightly-regulated expression of vax2, wherein both loss and increase in vax2 expression can result in aberrant closure. In an effort to explain the rarity of superior coloboma, we are currently testing the hypothesis that multiple loci are mutated. We have recently identified a second variant in patient #4, predicted to strongly reduce the function of the planar cell polarity gene, SCRIBBLE. Preliminary zebrafish experiments demonstrate a key role for planar polarity in SOS closure. Thus, our results provide support for a model in which multiple pathways are essential for SOS closure, including both DV axis specification and planar cell polarity.