Pax2 positively regulates angiogenesis in order to initiate optic fissure fusion.

Abstract

Purpose : Organogenesis of the vertebrate eye is intimately tied to the process of epithelial sheet fusion (ESF). Formation of a hemispherical eye involves a naturally occurring ventral gap, or optic fissure. Fusion of this fissure requires timely execution of epithelial sheet fusion as its failure results in a congenital blinding disorder Coloboma. Recent studies have linked endothelial vasculature precursor cells as potential triggers for optic fissure ESF initiation. In order examine this link, we analyzed the basic molecular events associated with ESF and haloid vascularization in a zebrafish Pax2^noi coloboma model.

Methods : ESF was examined using immunohistochemistry (IHC) in combination with confocal microscopy. Total RNA sequencing was performed on retinas isolated from Pax2^noi mutant embryos and the resulting data analyzed for regulators of angiogenesis. Retinal vascularization, tagged in the kdrl:mCherry transgenic line, was visualized in real-time confocal microscopy. Modulation of pax2 and angiogenesis regulators was achieved using mRNA or morpholino injections.

Results : Our data indicates that ESF in the optic fissure initiates at ~ 36hpf and is preceded by an accumulation of polymerizing F-actin. Fusion concludes by ~72-80hpf. In contrast, the accumulation of F-actin and the disassembly of the basement membrane are deficient in Pax2^noi embryos, which correlates to the persistence of the fissure and subsequent Coloboma. In order to examine how the ESF process is diminished in Pax2^noi embryos we compared retinal transcriptomic profiles between Pax2^noi and WT embryos. The resulting data uncovered a novel connection between regulation of angiogenesis and fusion. ADAMTS1, an anti-angiogenic factor, was found to be up-regulated ~2 fold, in the absence of Pax2. We also observed a ~200 fold reduction in the expression of talin1, a known regulator of endothelial vasculature migration. Imaging of retinal vascularization indicated a deficit in the Pax2^noi embryos while pharmacological inhibition of VEGF using DMH4 phenocopied the pax2^noi phenotype. Furthermore, injection of ADAMTS1 mRNA inhibited retinal vascularization and prevented fissure fusion.

Conclusions : Taken together, we propose that Pax2 negatively regulates ADAMTS1 expression in order to time the vascularization of the retina which in turn directly signals to initiate fissure fusion via cytoskeletal rearrangements and subsequent BM remodelling.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.