Pax2a Regulates Endothelial Cell Invasion into the Optic Fissure Resulting in Degradation of the Basement Membrane by mmp2

Abstract

Epithelial Sheet Fusion (ESF), a process by which two populations of epithelial cells fuse to create a continuous sheet, is a crucial aspect of embryogenesis. ESF can be observed in the development of many tissues, including fusion of the optic fissure (OF) during retinal development in vertebrates. Failure in fusion of the OF results in congenital coloboma, a leading cause of pediatric blindness. Unfortunately, majority of coloboma cases lack a definitive genetic cause. This is likely reflected by the fact that the molecular mechanisms driving OF ESF are still poorly understood. This study aims to identify the molecular machinery regulating ESF during OF fusion in zebrafish. To do so, we utilized a well characterized mutant line of zebrafish carrying a mutation in the Pax2a gene. Pax2a is a transcription factor known to be required for OF fusion and has been linked to cases of coloboma in many species. The mutant pax2anoi/noi line of zebrafish display a completely penetrant coloboma phenotype. To assess transcriptional differences between normal and abnormal OF fusion, RNA sequencing was performed comparing WT and pax2anoi/noi whole eye transcriptomes. The results indicated a down‐regulation of hyaloid vasculature regulators. Examining retinal vasculature in Tg[kdrl:mCherry], pax2anoi/noi mutants revealed a significant reduction and malformation of the hyaloid vasculature. Subsequently, OF fusion was analyzed in the absence of vasculature. Treatment with a pharmacological inhibitor of VEGF signaling, DMH‐4, completely inhibited the establishment of hyaloid vasculature in the retina. In the presence of DMH4, the OF failed to fuse. In order to understand the mechanism behind the reduction of vasculature in pax2noi/noi embryos we returned to the transcriptomic data and analyzed for angiogenic regulators. This analysis identified adamts1, an anti‐angiogenic protease which sequesters VEGF ligands, to be up‐regulated in pax2noi/noi embryos. As validation of our findings, overexpression of adamts1 mRNA in WT embryos successfully phenocopied the pax2noi/noi associated failure of OF fusion. Lastly, in order to determine the functional role of hyaloid vasculature cells during OF fusion we examined basement membrane remodeling. Matrix metalloproteinases (MMP’s) have the ability to degrade the BM. After characterizing expression of all zebrafish mmp’s, mmp2, mmp14a and mmp14b were found to be expressed in the OF. Using two‐color in‐situ hybridization we confirmed that mmp2 and mmp14 expression in OF is confined to the hyaloid vasculature cells. Furthermore, mmp2 and mmp14 expression is decreased in the OF in both DMH‐4 treated and Pax2 mutant embryos. Treatment with a pharmacological inhibitor specific to mmp2, ARP101 resulted in a failure of OF BM remodeling and subsequently fusion. Overall, our results show that pax2a negatively regulates adamts1 expression to enable proper vascularization of the developing retina. Vascularization is subsequently necessary for optic fissure fusion as hyaloid vasculature cells provide mmp2 necessary for the degradation of the OF BM and thus OF fusion.Support or Funding InformationLyman T. Johnson Fellowship, Knights Templar Career Starter Grant