Pax2a regulates angiogenesis to facilitate fusion of the optic fissure

Formation of the vertebrate retina involves timely and precise fusion of the optic fissure, failure of which results in the congenital blinding disorder coloboma. The optic fissure forms in consequence of optic cup morphogenesis and has a functional role as a conduit for migration of endothelial vasculature during hyaloid vasculogenesis. Recent studies have linked hyaloid vasculature precursor cells to be potential triggers, initiating optic fissure fusion. In order to examine this link, we analyzed the molecular events associated with optic fissure fusion and hyaloid vasculogenesis in the zebrafish pax2a mutant coloboma model. By means of wholemount immunohistochemistry, we determined that optic fissure fusion initiation, as indicated by laminin remodeling, is preceded by a spike in F-actin signaling. In contrast, the spike in F-actin activity and the disassembly of the basement membrane (BM) were misregulated in pax2a-/- embryos. To examine how the fusion process is disrupted in the absence of pax2a function, we compared transcriptomic profiles between pax2a-/- and wildtype (WT) embryos. This analysis uncovered a novel connection between regulation of angiogenesis and fusion. Loss of pax2a function resulted in significant reduction of talin1 expression, a known regulator of endothelial vasculature migration associated with fissure fusion. In addition, loss of pax2a also 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 pax2a-/- embryos while pharmacological inhibition of VEGF using DMH4 or ADAMTS1 mRNA overexpression phenocopied the pax2a mutant phenotype. Taken together, we propose that Pax2a positively regulates tln1 while negatively regulating ADAMTS1 expression. This enables timely hyaloid vascularization of the retina which in turn directly signals to initiate fissure fusion via cytoskeletal rearrangements and subsequent BM remodelling.