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Nicholas Carrara, Jakub Famulski; NIDOGEN: A LYNCHPIN OF BASEMENT MEMBRANE DISASSEMBLY PRIOR TO CHOROID FISSURE FUSION.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):326. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In the developing embryo, the fusion of two opposing epithelial sheets into one uniform layer denotes the completion of many developmental events. Failure of this epithelial sheet fusion event (ESF) within the choroid fissure (CF) is associated with the congenital disorder Ocular Coloboma and is one of the leading causes of pediatric blindness. A requirement for a coordinated dismantling of the basement membrane (BM) to allow for this fusion event to occur is undoubted, however the underlying mechanisms involved in this process are poorly understood. Due to its well characterized crosslinking capabilities and architectural importance, we have begun to investigate the functional role of nidogen (nid) during assembly and disassembly of the CF BM.
To investigate the function of nid and the core BM components during ESF we have employed the zebrafish model to perform a whole mount in situ hybridization expression analysis and fluorescent immunohistochemistry for all 4 major BM components during CF fusion. To analyze the functional contribution of nid to CF fusion, we are using both translation blocking morpholinos and dominant negative nid constructs. Lastly we have combined our loss of function approach with real-time in vivo microscopy to visualize the consequences of eye development in the absence of nid.
Our WISH analysis has revealed that the expression of 3 nid orthologues (1a, 1b and 2a) decreases prior to that of other BM components. Additionally, we have generated preliminary IHC data tracking the disassembly of core BM components prior to ESF. MO-mediated knock-down of nid1a and 1b, or the expression of dominant negative nid1b resulted in gross morphological, as well as BM organization defects in developing eyes. Nid1a/b morphant fish show a 24% decrease in eye size, as well as 25% increase in elongation of nasal-temporal axis. Moreover, live imaging of nid1a/b morphant eyes has captured the deviations that lead to these morphological changes in real-time.
Taken together, our these preliminary data suggest that the regulationstability of nid may plays a role in modifying the integrity of the BM in the eye and is therefore perhaps is a key regulator of its disassembly and subsequent CF fusion.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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