Uveal coloboma, a condition estimated to occur in ~1:10,000 live births, is a significant cause of blindness worldwide. It is characterized by a hole or cleft in the eye, and results from defective formation or closure of the optic fissure, a transient, yet vital structure through which retinal axons exit and vasculature enters the eye. Despite its importance, we have a poor understanding of the cellular and molecular mechanisms governing optic fissure development, especially the crucial initial step of formation: if the fissure does not form correctly, it will not undergo closure. The conserved Hedgehog (Hh) signaling pathway is crucial: in Gorlin Syndrome, overactive Hh signaling, in the context of the PTCH mutant (ptch2 in zebrafish) causes coloboma. In the past funding cycle, we determined the cell movements underlying optic fissure formation, using 4- dimensional imaging and cell tracking. Surprisingly, we found that a previously undescribed folding event in the ventral eye drives optic fissure formation, however, the underlying cell shape changes and rearrangements are unknown. In the ptch2 mutant, we uncovered key mechanisms by which overactive Hh signaling perturbs this process and found that downstream targets of Hh signaling are responsible for disrupting cell movements. Therefore, we performed single-cell RNA-seq to identify Hh target genes controlling optic fissure formation: matrix proteoglycans (agrin, dystroglycan, and syndecan-4) and mitochondria genes have informed our new directions. Here we take advantage of the unique optical transparency and rapid development of zebrafish embryos to directly examine optic fissure formation in vivo. We recently developed imaging and computational approaches to visualize and quantitatively analyze dynamics of cell movements, shape, and orientation driving optic fissure formation for the first time, as well as molecular genetic methods to perturb signaling pathways. In this proposal, we will uncover the cellular mechanisms directly responsible for optic fissure formation, as well as the role of tissue-tissue interactions with the olfactory placode, and intrinsic metabolic activity. Our hypothesis is that active cell shape changes and reorientation drive optic fissure formation, and this process relies on mechanical coupling with the olfactory placode via matrix proteoglycans, as well as intracellular mitochondrial dynamics. Combining molecular genetics, live imaging, custom computational methods and quantitative image analyses, we will test this hypothesis in the following specific aims: (1) establish the morphogenetic mechanisms driving optic fissure formation and its disruption in coloboma; (2) determine the cellular and molecular mechanisms by which the olfactory placode influences optic fissure formation; and (3) determine how mitochondrial dynamics contribute to optic fissure formation and its disruption in coloboma. Our proposed mechanistic experiments will expand the coloboma gene regu...