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Kristen Kwan, Emily Wirick; Hedgehog signaling regulates cell movements underlying choroid fissure formation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):710.
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© ARVO (1962-2015); The Authors (2016-present)
The choroid fissure is a transient, yet critical structure through which retinal axons exit and vasculature enters the eye. Disruption of choroid fissure formation or fusion results in uveal coloboma. Loss-of-function mutations in the Hedgehog (Hh) receptor patched2 result in colobomata in humans and zebrafish. These mutations result in overactive Hh signaling, yet the specific mechanisms by which overactive signaling leads to colobomata are unknown. Previous work demonstrated that Hh signaling plays a key role in patterning the dorsal-ventral axis of the eye, however, it is unknown whether it plays a more direct role controlling cell movements during eye morphogenesis.
We developed 4-dimensional live imaging techniques for visualizing and tracking cell movements during optic cup morphogenesis. For cell tracking, embryos are labeled for membranes (EGFP-CAAX) and nuclei (H2A-mCherry); 4D datasets are acquired via laser scanning confocal microscopy. Cell tracking is performed using custom LongTracker manual tracking software, and results visualized using FluoRender. The photoactivatable fluorophore Kaede is used to quantify movements of specific cell populations contributing to the ventroanterior retina, including the anterior margin of the choroid fissure. Overactivation of Hh signaling is achieved via expression of activated Smoothened or use of the zebrafish patched2 (blowout) mutant.
We find that cells moving into the optic vesicle during a specific time period that we term late evagination populate the ventroanterior retina, including the anterior margin of the choroid fissure. We hypothesized that activated Hh signaling would affect late evagination, thereby disrupting choroid fissure formation. We find that activated Hh signaling, either via expression of activated Smoothened or using the zebrafish patched2 mutant, inhibits late evagination cell movements: cells do not migrate out of the brain, and as a result, choroid fissure formation is disrupted, resulting in colobomata.
We have identified the cellular origin of the anterior choroid fissure. Overactive Hh signaling affects a very early step of optic cup and choroid fissure formation, suggesting a direct effect on morphogenesis and cell behavior. We are currently determining the effect of overactive Hh signaling on actin-based single-cell behaviors, as well as whether Hh is acting via canonical or non-canonical pathways.
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