Comprehensive Analysis of Periocular Mesenchyme Composition, Specification and Function During Anterior Segment Formation

Inherited congenital anterior segment dysgenesis (ASD) disorders are a major cause of vision impairment and blindness. They include developmental ocular deficiencies of the iris, cornea and lens in addition to significant predisposition, ~50%, to glaucoma. From various studies in recent years it has become clear that many congenital disorders and predispositions arise from early developmental defects, both major and subtle. Examining the developmental process giving rise to these essential components of the visual system is therefore a fruitful approach to studying the disease. The anterior segment derives largely from the early migrating neural crest cells that target specifically to the anterior of the developing retina and are loosely classified as periocular mesenchyme (POM). POM deficiency has been demonstrated in certain cases of ASD. I believe a major roadblock in efforts to treat and understand ASD has been the limited examination of POM biology and function during early ocular development. This hampers efforts to characterize the etiology of disease and screening in ASD families, in addition to delaying attempts at iPSC-mediated approaches for treatment of ASD. To circumvent these problems, we aim to comprehensively characterize POM cell specification, migration, targeting, differentiation, and ultimately contribution to anterior segment dysgenesis. This is the next logical and necessary step in order for the field to continue the evolution of therapy and screening for glaucoma and ASD disorders. Our immediate goal is to test the hypothesis that POM cells delineate into various subpopulations and specify to become the anterior segment mesenchyme (ASM). To test our hypothesis we propose to employ the highly versatile zebrafish embryo model system and comprehensively examine POM cell development while cataloging their contribution to the anterior segment through genetic, molecular and cutting edge in vivo imaging approaches. Our specific aims are: Aim 1: Characterize specification and define the composition of the ASM the using a combination of gene expression analysis paired with light-sheet in vivo time lapse imaging and confocal microscopy. Aim 2: Track the migratory behavior and lineage trace distinct populations of ASM during anterior segment formation using light-sheet in vivo time lapse imaging and confocal microscopy paired with a novel fate mapping optogenetic tracing mechanism. Aim 3: Generate a comprehensive ASM transcriptome for screening novel targets in ASD patient samples using a combination of flow cytometry coupled to RNAseq and next generation exome sequencing.