Optic cup morphogenesis is the process by which the nascent optic vesicle undergoes complex cell and tissue movements to transform into the optic cup—proper structure of which is critical for visual function. Coloboma is a structural defect of the eye arising from improper development of the choroid fissure—a transient yet critical optic cup structure through which retinal axons exit the eye and vasculature enters the eye. The Hedgehog (Hh) signaling pathway is a major effector of choroid fissure development: loss of function mutations in the Hh receptor patched, which lead to overactive Hh signaling, result in coloboma in zebrafish and humans. Despite the importance of the choroid fissure, we have a poor understanding of the cellular mechanisms underlying its morphogenesis.
Here we combine multidimensional timelapse imaging with custom cell tracking software to establish the cell movements underlying choroid fissure formation in zebrafish. Specifically, we mapped the origin and trajectories of cells of the nasal and temporal choroid fissure margins under wild type conditions, and we determined the precise movements that are disrupted in patched2tc294z homozygous mutants. Our results indicate that cells that should be destined for the nasal margin of the choroid fissure stall in their migration, rendering them stuck in the optic stalk. To examine the migration defect at the cellular level, we marked cells using the photoactivatable fluorophore Kaede: in wild type embryos, cells exhibit a bipolar morphology while moving to form the choroid fissure. In patched2 mutant embryos, these cells fail to maintain their initial bipolar morphology: they take on an aberrant multipolar morphology and cease movement within the optic stalk region. We conclude that overactive Hh signaling causes coloboma by disrupting cell movements underlying formation of the choroid fissure.
Having defined the cellular events underlying choroid fissure formation in wild type and patched2 mutant embryos, we set out to determine the molecular mechanisms by which loss of patched2 alters cell behavior. First, we asked in which cells overactive Hh signaling acts to disrupt choroid fissure cell movements. Surprisingly, using blastula cell transplantations, we find evidence for a non-cell autonomous role for overactive Hh signaling in controlling cell migration and morphology. This suggests interaction between migrating cells and the patched2 mutant environment. We are now determining whether overactive Hh signaling due to loss of patched2 is acting via canonical or non-canonical downstream signaling pathways to disrupt cell morphology and migration.