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Kristen Kwan, Chase Bryan, Macaulie Casey, Rebecca L Pfeiffer, Bryan W Jones; Migratory neural crest directs optic cup morphogenesis by depositing nidogens to build extracellular matrix superstructure. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6017. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Although the neural crest contributes to mature eye structures, its role in controlling early stages of eye development, specifically optic cup morphogenesis, is poorly understood. In mouse, removal of neural crest leads to optic cup defects, but the underlying cellular and molecular mechanisms are unknown. Using 4-dimensional live imaging and zebrafish molecular genetics, we set out to determine precisely how neural crest regulates optic cup formation. In the absence of neural crest, what cell movements are disrupted? What molecule(s) is neural crest providing to control optic cup morphogenesis?
4D imaging datasets of optic cup morphogenesis are acquired via confocal microscopy. To test the role of neural crest, we use tfap2a;foxd3 double mutants. Neural crest migration is visualized using transgenic embryos (sox10:mRFP or GFP). LongTracker is used for 4D cell tracking; results are visualized using FluoRender and ImageJ. Transmission electron microscopy is used to image basement membranes.
We find that neural crest cells migrate and enwrap the RPE side of the developing optic cup. Loss of neural crest results in defective optic cup invagination: cell movement around the optic vesicle rim, from the medial layer to the retinal layer, is specifically impaired. Electron microscopy data indicate that basement membrane assembly is lost in the absence of neural crest, but only around the RPE, where neural crest is normally in contact with the eye. Expression of individual ECM components is unaffected, indicating a specific defect in ECM assembly. Nidogen, an extracellular matrix crosslinking protein essential for optic cup morphogenesis in ES cells, appears to be crucial: dominant negative nidogen disrupts wild type optic cup morphogenesis, and overexpression of wild type nidogen partially rescues optic cup morphogenesis in the neural crest mutant.
Our data suggest that during optic cup formation, migrating neural crest cells enwrap the developing eye and drive the cell movements underlying optic cup invagination. These neural crest cells deposit nidogens around the RPE to build extracellular matrix superstructure, which can support proper cell movements. We are currently generating nidogen quadruple mutants to examine the full loss-of-function phenotype in the eye.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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