The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye

Abstract

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, β-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Zebrafish mab21l2 mutants possess severe defects in optic cup morphogenesis, lens and cornea development

“…mab21l2 −/− mutants possessed colobomas of varying severity, ranging from very severe retinal blowouts, where retinal and RPE tissue extruded out the back of the eye, to mild discontinuities of the retinal lamina visible only in sectioned tissue. Choroid fissure closure is a precisely orchestrated process, and critical events include specification of the optic stalk/optic cup boundary, tissue growth and morphogenesis, and basement membrane breakdown . The optic stalk/optic cup markers pax6 and pax2 were correctly distributed in mab21l2 −/− embryos, leading us to conclude that early tissue patterning defects are unlikely to be responsible for colobomas.…”

“…Alternatively, it is known that choroid fissure closure requires contributions from the hyaloid vasculature, neural‐crest derived periocular mesenchyme, and the retina itself . Of these tissues, mab21l2 is known to be highly expressed in the retina, in a domain that gradually restricts to a small region of cells that comprise the lateral edges of the choroid fissure .…”

“…It is also possible that mab21l2 function is associated with cell nonautonomous signals that modulate the activities of other cells types at or near the choroid fissure that are required for proper closure. With respect to cell nonautonomous functions, it is interesting that the laminin α1‐containing basement membrane was not properly degraded in the mab21l2 −/− choroid fissure, given that previous studies have shown that the hyaloid vasculature contributes to the breakdown process, but is not the sole requirement . Indeed, retinal cells themselves extend protrusions into the site of membrane breakdown, although it is not known whether these are functionally relevant.…”

“…mab21l2 −/− mutants with severe colobomas showed extrusion of retinal and RPE tissue out of the eye (Figure 4E, arrow). In milder mab21l2 −/− mutants, sagittal sections revealed modest colobomas, detected by the misalignment of the retinal layers in the ventral portion of the It is known that breakdown of the basement membrane that lines the two edges of the retina/RPE within the choroid fissure correlates with choroid fissure closure, and that failure of this process can lead to colobomas, [37][38][39][40][41] We investigated whether basement membrane breakdown proceeded correctly in mab21l2 −/− mutants by means of immunohistochemical staining for laminin α1, a component of laminin-111, which is a constituent of the basement membrane lining the edges of the choroid fissure, 42,43 comparing wild-type to mutant eyes at 2 dpf, when most wild-type fish have completed choroid fissure closure. In wild-type embryos, the laminin α1 distribution around the outside of the fused eyecup is continuous, which indicates successful closure and fusion of the choroid fissure 44 and no laminin α1 is detected at the site of the now fused choroid fissure ( Figure 4G,G 0 , arrowheads).…”

The occhiolino (occ) mutant Zebrafish, a model for development of the optical function in the biological lens

“…In the developing lens, the capsule is a basement membrane for the attachment of the basal surface of epithelial cells during the synchronized proliferation, migration, and elongation to form layers of transparent lens fibers organized precisely into the optical element of the Zebrafish visual system. As a key component of the basement membrane, laminin is a marker for the lens capsule (Parmigiani and McAvoy, ; Menko et al, ; Norose et al, ; Yan et al, ; Lee and Gross, ; Danysh and Duncan, ; Kwan, ; James et al, ; Patel et al, ). Laminin mutants disrupt basement membrane structure, resulting in abnormal lens development in Zebrafish as early as 3 dpf, with some mutations causing complete loss of the lens as early at 7 dpf (Parmigiani and McAvoy, ; Semina et al, ; Zinkevich et al, ; Pathania et al, ; Patel et al, ).…”