Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (1~ in 5,000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggest multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicates that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased RPE proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during optic fissure closure
Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (1~ in 5,000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggest multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicates that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased RPE proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F--actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during optic fissure closure.
We explored the mechanisms and regulation of rhodopsin endocytosis in Drosophila by examining the trafficking itineraries of the major rhodopsin (Rh1) in vivo using transgenic flies expressing a modified Rh1 containing a C‐terminal mCherry tag (Rh1‐mCherry). By fluorescence microscopy, Rh1‐mCherry was detected in the visual organelles (rhabdomeres) of photoreceptors. Upon continued light stimulation, Rh1‐mCherry display light‐dependent endocytosis, and becomes enriched in the cytoplasm of photoreceptors. Internalization of rhodopsin may be mediated by the clathrin‐mediated endocytosis (CME). CME requires the presence of a minor phospholipid, phosphoinositol‐4, 5‐bisphosphate (PIP2) in the membrane. Interestingly, perturbation of the PIP2 biosynthesis fails to prevent the endocytosis of Rh1‐mCherry in rdgA mutants. The rdgA gene encodes diacylglycerol kinase critical for the recycling of PIP2.We also explored the contribution of two visual arrestins, Arr1 and Arr2, as adaptor proteins in promoting the endocytosis of Rh1. We introduced Rh1‐mCherry into either arr1 or arr2 mutant background and investigated how trafficking is affected. We show that internalization of Rh1‐mCherry is drastically reduced in the arr2 mutant. Arr2 is expressed 7fold more abundant than Arr1. Based on the finding, we conclude that Arr2 is critical for orchestrating the light‐dependent endocytosis of Rh1‐mCherry. Importantly, this Arr2‐mediated event does not appear to belong to the classical CME as it is insensitive to a reduction of PIP2 in the membrane.
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