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Andrea James; p190RhoGAP is Required for Proper Temporal Completion of Chroid Fissure Closure in the Zebrafish Eye. Invest. Ophthalmol. Vis. Sci. 2018;59(9):323.
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© ARVO (1962-2015); The Authors (2016-present)
A critical aspect of vertebrate eye development is closure of the transient structure, the choroid fissure (CF). Defects in CF closure result in colobomas, which contribute to 3-11% of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, there is limited understanding of the cell biological underpinnings of CF closure. Here we utilize the zebrafish embryo to identify the molecular signaling required for the proper CF closure. Previously, we demonstrated a transient, 2 hr, βcatenin and actin fusion seam in the sealing RPE/retina tissue of the CF indicating a possible role for classical cadherin based adherens junction formation during the final stages of CF closure. We hypothesize CF fusion utilizes a classical cadherin to regulate actin dynamics in a p190RhoGAP dependent manner.
To verify the localization of classical cadherins during CF closure, wildtype zebrafish embryos were collected pre-, during and post-CF closure and cryosections prepared for subsequent confocal immunofluorescence imaging and a spatiotemporal analysis in the CF in regards to β-catenin, actin and cadherin localization. To identify the role of actin regulation during CF closure, a p190RhoGAP TALEN mutant was created and embryos imaged for general phenotype 2-6 dpf. Mutant and sibling embryos were similarly cryosectioned and imaged for spatiotemporal analysis in the CF as above.
Cadherin immunoflouorescence demonstrates localization in the CF in wildtype embryos similar to the b-catenin/actin “fusion seam”. When confirming the p190RhoGAP TALEN mutant zebrafish line we identified possible microencephaly, reduction in jaw formation and lack of a swim bladder in mutant embryos at 5 dpf. CF closure initiated at normal times in p190RhoGAP mutant in comparison to wildtype. Further analysis demonstrated the p190RhoGAP mutant fusion seam was present 8 hours later despite sibling embryos fully completing CF closure.
Our data indicate the transient nature of the fusion seam may be regulated in a cadherin dependent manner. In addition, data from our p190RhoGAP TALEN mutant demonstrate a role for proper regulation of the actin regulator RhoGTPase. This disruption of the transient nature of the CF fusion seam may lead to dysfunction in retinal development, Further work is needed to determine how timing of CF closure is required for retinal differentiation.
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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