Author ORCID Identifier

https://orcid.org/0000-0002-6787-8040

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Biology

First Advisor

Dr. Jakub Famulski

Abstract

Vertebrate retinal development requires timely and precise fusion of the optic fissure. Failure of this event leads to congenital vision impairment in the form of coloboma. Recent studies have suggested hyaloid vasculature to be involved in OF fusion. In order to examine this link, we analyzed optic fissure fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a-/- embryos fail to accumulate F-actin in the optic fissure prior to basement membrane (BM) degradation. Furthermore, using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a-/- embryos. When examining the connection between pax2a loss of function and hyaloid vasculature, we observed significant reduction of talin1 expression, a regulator of hyaloid vasculature. In addition, cranial VEGF expression was found to be reduced in pax2a-/- embryos. Pharmacological inhibition of VEGF signaling phenocopied the pax2a-/- vasculature, F-actin and BM degradation phenotypes. Lastly, we determined that optic fissure associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of optic fissure BM. We compared transcriptomic profiles between pax2a-/- and wildtype (WT) embryos. We sought to validate and investigate those targets which were thought to be of importance to the molecular machinery involved in fusion, particularly those involving the actin cytoskeleton and angiogenesis. This analysis uncovered a novel connection between regulation of angiogenesis and fusion. Loss of pax2a resulted in increased expression of an anti-angiogenic protease, ADAMTS1. 3D confocal and live imaging of retinal hyaloid vascularization in Tg[kdrl:mCherry] embryos indicated a significant deficit in ADAMTS1 mRNA injected embryos and phenocopied the pax2a mutant phenotype. We have also uncovered preliminary evidence that certain actin associated proteins are also differentially regulated, particularly rho-associated protein kinase (ROCK). Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF by negatively regulating ADAMTS1 expression. This enables the timely hyaloid vascularization of the retina which in turn directly signals to initiate fissure fusion via cytoskeletal rearrangements and providing the BM remodeler mmp2.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.021

Funding Information

National Eye Institute, R01 EY027805, 2018.

University of Kentucky Lyman T. Johnson Fellowship, 2015.

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