Stepwise Coordination of Eye Morphogenesis by Extracellular Matrix

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 DESCRIPTION (provided by applicant): Developmental defects in eye structure commonly account for visual impairment in newborns. Proper eye structure is initially established during the process of optic cup morphogenesis, during which the optic vesicle transforms into the optic cup via a series of complex cell and tissue rearrangements, with neural retina and retinal pigmented epithelium surrounding the newly formed lens. Recent advances in live imaging have begun to reveal the cellular processes underlying optic cup morphogenesis, yet molecular control of these critical events still remains largely unknown. A compelling candidate to play a role in controlling optic cup morphogenesis is the extracellular matrix (ECM), a complex, glycoprotein-rich layer that can regulate cell survival, movement, polarity, and tissue tension. ECM components, including laminin and type IV collagens, surround eye structures throughout optic cup morphogenesis in all vertebrates examined to date. Mutations in certain ECM components can lead to specific ocular pathologies in humans and model organisms, such as coloboma (failure of choroid fissure development) or lens defects. This suggests distinct requirements for ECM components during specific events in optic cup morphogenesis, yet this has not been examined in a systematic way. Zebrafish presents an ideal model system to study this process: optical transparency and rapid development offer a unique opportunity to directly watch eye formation in vivo. Further, we previously developed 4-dimensional imaging and computational techniques to track and visualize cell movements and behaviors throughout optic cup morphogenesis. This puts us in a unique position to quantitatively determine specific morphogenetic defects arising when particular matrix components are disrupted. In this proposal, we will determine the roles of laminin, type IV collagens, and the laminin-collagen cross-linking molecule nidogen during optic cup morphogenesis. I hypothesize that sequential steps of optic cup morphogenesis harbor distinct requirements for matrix components and higher-order assembly, and that these differential requirements underlie specific ocular pathologies seen in humans and model organisms. Combining molecular genetics with innovative 4-dimensional live imaging and computational methods, we will test this hypothesis in the following specific aims: (1) determine the role of matrix components and supramolecular assembly in choroid fissure formation; (2) determine how matrix components control optic cup invagination, taking advantage of a novel mutant, in which retina-lens de-adhesion (and possibly collagen signaling) are impaired; (3) determine the role of matrix components in establishing proper lens shape and ectoderm separation. The experiments proposed here will define the cellular and molecular dynamics of extracellular matrix adhesion underlying critical steps in optic cup morphogenesis, and the specific cellular functions executed by matrix during each step.