Hyaloid vasculature and mmp2 activity play a role during optic fissure fusion in zebrafish

Abstract

Vertebrate retinal development requires timely and precise fusion of the optic fissure (OF). 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 OF fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a −/− embryos fail to accumulate F-actin in the OF 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 OF associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of OF BM. Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF in order to facilitate availability of the BM remodeler mmp2. Ocular development is a highly conserved process amongst vertebrate species. Assembly of the hemispherical, retinal structure from an initially flat sheet of cells requires many complex morphogenetic movements. One such morphogenetic movement involves the invagination of the optic vesicle which results in a fissure forming at the ventral region of the developing retina. This fissure, known as the choroid or optic fissure (OF), enables hyaloid vasculature cell migration into the developing retina and subsequent establishment of the hyaloid vasculature. Hyaloid vasculature is a temporary circulatory system required for ocular development, and in most cases will degenerate once mature blood vessels begin to grow 1-4. As soon as the hyaloid vasculature has been established, the two opposing retinal epithelial sheets of the OF will undergo fusion. Thereby, they encase the ganglion cell axons localized in the optic stalk and complete retinal morphogenesis. Failure of OF fusion leads to a congenital blinding disorder known as coloboma 5-7. Coloboma is a prevalent cause of pediatric blindness, accounting for approximately 10% of cases worldwide 6,8. This makes it one of the leading causes of pediatric blindness. Coloboma is a spectrum disorder presenting unilaterally or bilaterally and ranging in severity from minor visual impairment, to complete blindness in the affected eye 9. This spectrum of severity is associated with the location and degree to which the OF was able to fuse and the severity of subsequent loss of ganglion cell axons 7. Coloboma has been studied for many decades in many different species. Work over this time has led to a general outline of the signaling and morphogen...

Ocular coloboma: Genetic variants reveal a dynamic model of eye development

“…This suggests that actin-based protrusions facilitate fissure closure and that cell movements and dynamics must be carefully regulated for choroid fissure to occur. A recent zebrafish study has shown that F-actin deposition correlates with the entry of the hyaloid vasculature in the eye, and inhibition of VEGF signaling (and thus ocular vascularization) results in loss of ocular tln1 expression, suggesting that these actin-based protrusions are derived from the developing ocular vasculature (Weaver, Piedade, Meshram, & Famulski, 2020).…”

“…Similarly, cloche mutants, which lack all endothelial cells, also have delayed choroid fissure closure. It has been suggested that blood vessels themselves secrete signals that promote basement membrane breakdown and choroid fissure closure; endothelial cells have been shown to secrete ECM remodeling proteins such as matrix metalloproteinases, therefore it is plausible that blood vessels play an active role in breaking down the basement membrane surrounding the fissure prior to closure; a recent study performed in zebrafish showed that the endothelial cells of the hyaloid system produce and secrete Mmp2, a matrix metalloproteinase that has well‐characterized roles in extracellular matrix breakdown and remodeling (Weaver et al, 2020). Pharmacological inhibition of mmp2 results in choroid fissure closure defects, suggesting that mmp2 production by the hyaloid is an essential step of choroid fissure closure (Weaver et al, 2020).…”

Loss of zebrafish dzip1 results in inappropriate recruitment of periocular mesenchyme to the optic fissure and ocular coloboma

“…The POM has been implicated in optic fissure closure [ 15 , 65 – 68 ]: electron microscopy revealed POM cells near sites where basement membrane breakdown were taking place and transplanting the eye to sites distant from POM led to failure of optic fissure closure [ 11 , 14 , 69 ]. Further, recent work has proposed that POM may be a source of matrix metalloproteinases to degrade basement membrane [ 70 ]. Therefore, disruption to POM localization could be a cause of optic fissure closure defects, and therefore coloboma, in the dzip1 mutant.…”

Closing the Gap: Mechanisms of Epithelial Fusion During Optic Fissure Closure

“…Another class of proteolytic enzymes implicated in OFC are matrix metalloproteases (MMPs), such as those recently identified as secreted from developing hyaloid vasculature cells migrating between the zebrafish fissure margins (mmp2, 14a, 14b) and in transcriptomic studies (mmp23b) ( Richardson et al, 2019 ; Weaver et al, 2020 ). Hyaloid vasculature in zebrafish is necessary for OF BM degradation, as genetic, chemical, or its physical perturbation results in a persistent BM and prevents correct OFC ( Gestri et al, 2018 ; Weaver et al, 2020 ). Significantly, mmp2 was specifically implicated as functionally necessary for BM degradation in the optic fissure ( Weaver et al, 2020 ).…”

“…Hyaloid vasculature in zebrafish is necessary for OF BM degradation, as genetic, chemical, or its physical perturbation results in a persistent BM and prevents correct OFC ( Gestri et al, 2018 ; Weaver et al, 2020 ). Significantly, mmp2 was specifically implicated as functionally necessary for BM degradation in the optic fissure ( Weaver et al, 2020 ). How these findings in zebrafish OFC translate to other vertebrates isn’t clear, and orthologs of MMPs acting in OFC in other species (or human colobomas) have not yet been identified.…”