Examining the Critical Role of Nidogen in Ocular Morphogenesis, Retinal Fissure Fusion and Coloboma

The importance of accurate formation of the visual system, in particular retinal morphogenesis, is clearly reflected in congenital ocular disorders. One of the leading causes of pediatric blindness, coloboma, results from incomplete fusion of retinal tissue during early embryonic development. In my Career Starter proposal we aimed to examine the molecular mechanisms of this critical event by analyzing basement membrane (BM) remodeling in zebrafish embryos. As part of this effort we have begun to characterize the function of core BM components, in particular nidogen. Previously uncharacterized in zebrafish, expression patterns of 4 zebrafish nidogen orthologues were analyzed and the results confirmed the presence of nidogen in the developing retina. In particular, Nidogen 1a, 1b and 2a were found to express within the optic and superior fissures during early retinal morphogenesis. To analyze nidogen loss of function phenotypes we examined nidogen 2a mutants in combination with nidogen 1a and 1b morpholinos. Our loss of function approach revealed a critical requirement of nidogen in retinal BM assembly as well as accurate retinal morphogenesis. Loss of function for either nidogen 1a or 1b resulted in misshapen and smaller retinas and BM defects. Serving as a cross linker between core BM components laminin, perlecan and collagen, and based on our preliminary data, we hypothesize that nidogen acts as a linchpin required to maintain integrity of retinal fissure BM. Along those same lines we also predict that nidogen represents a likely target during BM remodeling required for triggering optic fissure fusion. To test our hypothesis we propose to undertake the following: AIM I: Examine the role of nidogen and basement membrane integrity during retinal morphogenesis. Combining cutting edge molecular tools for genomic engineering with real-time light sheet microscopy we seek to visualize the consequences of BM disruption in developing zebrafish embryos. To extend our analysis past retinal morphogenesis we propose to examine expression of retinal patterning genes as well as document photoreceptor development in the absence of nidogen function. AIM II: Determine whether nidogen acts as a lynchpin during disassembly of retinal fissure BM. Using a combination of immunohistochemistry, fluorescence imaging, and gene expression analysis we aim to decipher the exact timing and order of basement membrane disassembly during optic and superior fissure fusion. Our established nidogen loss of function models will serve to analyze the functional contribution of nidogen during fissure fusion while gain of function approaches will assay whether nidogen is directly targeted for degradation. Lastly we aim to identify the remodeling enzymes directly involved in nidogen-based BM remodeling. We have recently identified MMP9, MMP24 and ADAMTS6 as potential nidogen proteases expressed in the retina and fissure and will test their functional roles in initiating fissure fusion. Ultimately we aim to correlate the functional contribution of the BM during ocular morphogenesis and the molecular mechanisms regulating BM disassembly during fissure fusion to congenital ocular blinding disorders, in particular coloboma.