Investigation of the Genetic Etiology of Superior Coloboma and the Formation and Closure of Superior Ocular Sulcus

Abstract

Congenital ocular coloboma is a rare genetic disorder that is typically observed as a cleft in the inferior aspect of the eye due to failed choroid fissure closure during embryogenesis. Together with microphthalmia (small eyes) and anophthalmia (absence of eyes), it represents 3-11% of childhood blindness worldwide. In addition to environmental factors, genetic analyses of coloboma patients have revealed many key eye development genes associated with ocular coloboma, involved in regulation of initial specification of the eye field, migration of retinal precursors, patterning of the retina, neural crest cell biology, and activity of head mesoderm. In this thesis, we describe individuals with coloboma in the superior aspect of the iris, leading 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. Through our analysis of patient exome-sequencing data and the use of zebrafish, we investigate the roles of dorsal eye patterning genes, BMPR1A and TBX2, and determine that aberrant dorsal eye patterning results in SOS closure defects, and that the SOS acts as a conduit for blood vessels. Additionally, I investigate the role of another key dorsal-ventral (DV) eye axis patterning gene, VAX2, a transcription factor regulated by Shh signaling that is expressed in the ventral eye. I observe that maternal zygotic homozygous vax2-null embryos display SOS closure delay. In addition, overexpression of human wildtype VAX2 mRNA in zebrafish embryos leads to SOS closure delay and perturbed expression of DV eye 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 requires a tightly-regulated expression of vax2, wherein both loss and increase in vax2 expression can result in improper closure. Subsequently, I present a brief investigation of the role of TSC2, a key regulator of mTOR signaling, on SOS closure. Through a morpholino-based knockdown approach, I establish that loss of tsc2 in zebrafish results in SOS closure delay, thus identifying the mTOR signaling pathway as a possible regulator of SOS closure during eye development, independent of DV eye axis patterning. Finally, in an effort to expand our understanding of the genetic etiology of superior coloboma and the genetic factors that regulate SOS formation and closure, I present an RNA-Seq data set generated from our models of SOS closure delay. Overall, this thesis aims to identify genetic factors that regulate the SOS to reveal the etiology of superior coloboma, a disorder of which there is very little known. In addition, these studies of the SOS add to the current understanding of eye development as a whole, as we begin to elucidate the role of this novel developmental structure during early embryogenesis.