The long-term goal of our research is to elucidate the mechanistic basis of ocular morphogenesis in vertebrates. During ocular morphogenesis, the optic primordia undergo a complex series of morphogenetic movements that ultimately result in a bilayered optic cup containing the prospective retina and RPE. The neuroectodermal layers of each optic primordium must fuse along its proximo-distal axis such that the retina and RPE will be confined within the limits of the optic cup during the early phases of ocular morphogenesis. Fusion occurs along a distinct region of the optic cup called the choroid fissure. The molecular mechanisms and the cellular processes that facilitate choroid fissure closure have not been well characterized in any vertebrate organism. Defects in choroid fissure closure result in chorioretinal colobomas, debilitating congenital malformations of the human eye. The molecular basis of the defects underlying chorioretinal colobomas is largely unknown and there are few animal models for studying their etiology. Experiments in this proposal will utilize the zebrafish, Danio rerio, to elucidate the molecular and cellular mechanisms that underlie ocular morphogenesis and choroid fissure closure. The results of these studies will be significant, as they will begin to shed light on the molecular and morphogenetic defects that lead to chorioretinal colobomas in humans. (Specific Aim 1) We will determine the molecular, cellular and morphogenetic bases for coloboma phenotypes in the zebrafish mutant blowout. We have cloned the disrupted locus in this mutant and identified a mutation in the patched1 gene, a negative regulator of the Hedgehog pathway. Here we will test the hypothesis that coloboma phenotypes in blowout result from overproliferation of the optic stalk and we will determine the molecular mechanisms underlying this phenotype. (Specific Aim 2) Mutations in human PATCHED1 lead to Basal Cell Naevus Syndrome or Gorlin Syndrome (BCNS), a disorder that presents with ocular defects that include the formation of a glia-derived epiretinal membrane, retinal dysplasia and colobomas. The molecular and cellular basis of these defects is not known and we will utilize adult blowout/patched1 and leprechaun/patched2 mutants to test the hypothesis that the ocular defects in BCNS patients stem from unchecked proliferation of Muller glia within the retina that results from constitutive Hedgehog-dependent target gene expression. (Specific Aim 3) Downstream of the Hedgehog pathway, vax1 and vax2 occupy a key node in the gene regulatory network mediating ventral optic cup formation and choroid fissure closure. Utilizing a microarray approach we have identified over twenty genes whose expression in the ventral optic cup and choroid fissure is dependent on vax1 and vax2. In this Aim, we will focus on one specific vax1/vax2 target: integrin a5, a fibronectin-binding integrin that we hypothesize is required for choroid fissure cells to interact with a fibronectin-rich extracellular matrix within the fissure to stimulate its closure. We will utilize histological, molecular and in vivo imaging techniques to test this hypothesis.
The research proposed in this grant will shed light on the molecular, cellular and morphogenetic underpinnings governing choroid fissure closure. In humans, defects in choroid fissure closure result in chorioretinal colobomas: congenital ocular defects where large regions of the eye lack both retina and retinal pigmented epithelium because these structures failed to be contained within the eyecup during ocular morphogenesis. Identification of factors required for choroid fissure closure will have a direct impact on our understanding of coloboma formation and on the development of possible treatments.
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