Pax2a regulates angiogenesis to facilitate fusion of the optic fissure

ABSTRACT 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. AUTHOR SUMMARY Formation of a fully functioning human eye requires precise and coordinated cellular mechanisms during very early development. One such mechanism is responsible for the formation of the eyeball structure from a flat sheet of cells to a hemispherical organ. In order to facilitate the biophysical properties of this shape change, an opening on the ventral side of the eye forms and must be closed in order to properly form the eyeball. Failure of this event leads to pediatric blindness in the form of a disease termed Coloboma. While it has been studied by researchers for almost a century, we still lack a significant understanding of how and why some children are born with this disease. In this study, we aimed to better understand how the closure of this opening is triggered by the developing tissues. What we found is, that non-eye cells which are found within this opening and are making vasculature networks to nourish the eye, are likely triggers for the fusion event. We found that regulating their migration and proliferation, also known as angiogenesis, is required for closure of this opening and therefore proper development of the eye.