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. 2016 Nov 15;419(2):262-272.
doi: 10.1016/j.ydbio.2016.09.008. Epub 2016 Sep 12.

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

Andrea James #  1   2 , Chanjae Lee #  1 , Andre M Williams  1 , Krista Angileri  1   2 , Kira L Lathrop  2   3 , Jeffrey M Gross  1   2   4
Affiliations

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

Andrea James et al. Dev Biol. .

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.

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Figures

Figure 1
Figure 1. Temporal and spatial dynamics of basement membrane breakdown during choroid fissure closure in zebrafish
(A) Schematic depicting the approximate level of sections in B-M along the proximal-distal axis of the CF. The vitreous cavity was defined as central, and sections were taken at 12um intervals proximally and distally from this point. (B-M) Sagittal sections along the proximal-distal axis of the retina, immunostained for Lam-111 expression. (B-E) 31 hpf, (F-I) 34 hpf, (J-M) 36 hpf. Insets in D, H show high magnification views of the regions in the dashed boxes. (N) Schematic depicting the plane of section for 48hpf embryos in O,P. (O,P) Representative sagittal sections along the proximal-distal axis of the retina immunostained for Lam-111. Scale bars = 20 μm.
Figure 2
Figure 2. in vivo imaging of choroid fissure closure in zebrafish
(A) Schematic depicting the approximate level of sections in B-D along the proximal-distal axis of the CF. The vitreous cavity was defined as central, and optical sections were taken at 16um intervals proximally and distally from this point. (B-D) membrane-GFP injected embryos were imaged throughout the CF. Single micron optical slices are shown from 44-49hpf and at three distinct proximal-distal regions of the CF. (B) Distally, the CF remains open until at least 49hpf. (C) Distal/centrally, the CF appears to close between 46-47hpf. (D) Proximally, the CF already appears to be closed at 44hpf. Orange arrowheads in B,C mark open CF. White arrow in C marks what appears to be a closed CF. Dashed line outlines the RPE. Scale bar = 50 μm.
Figure 3
Figure 3. Temporal and spatial dynamics of tissue fusion during choroid fissure closure in zebrafish
Single micron optical sections from sagittal cryosections stained with phalloidin (green) and anti-β-catenin (red) at distinct proximal-distal regions of the CF over time. As in Figure 2, the vitreous cavity was defined as central, and sections were taken at 16um intervals proximally and distally from this point. (A-E) At 44hpf, the CF is fused in central/proximal sections (white arrow) based on co-localization between F-actin and β-catenin in a fusion ‘seam’. (F-J) At 45hpf, the two sides of the CF are tightly apposed but no fusion outside of the central-proximal region is detected. (K-O) At 47hpf, a fusion seam is present within central and proximal sections, and there are punctate regions of co-localization in distal/central sections. (P-T) At 49hpf, the fusion seam has disappeared in the central and proximal CF regions while it is appearing in the distal/central region. (U) Single 1um optical sections from one section plane aligned distal (left) to proximal (right) demonstrate a progressive co-localization of F-actin and β-catenin in the CF and formation of the fusion seam. Scale bar = 50μm (A-T) and 5μm (U).
Figure 4
Figure 4. Periocular mesenchymal cells contribute to CFC
(A-C) Sagittal views of the CF stained with anti-GFP (green), Lam-111 (red) and/or phalloidin (blue). (A) Few sox10:eGFP+ cells are detected in the CF, 37hpf section pictured. (B) fli1a:eGFP+ cells are retained in the CF. 36hpf section pictured. (C) fli1a:eGFP+ cells possess F-actin accumulations that localize to regions of BM breakdown. Arrows denote puncta of F-actin where Lam-111 is low or absent. 34hpf section pictured. Scale bar = 20μm (A,B) and 10μm (C).
Figure 5
Figure 5. talin1 is required for CFC in zebrafish
(A) talin1 is expressed within the POM and retinal/RPE cells lining the CF at 33hpf (arrow). (B,C) Lateral views of the eye of tln1hi3093Tg mutant (C) and wild-type sibling (B) at 3dpf. tln1 mutants possess colobomas. (D-F) Distal section through the eye of a 32hpf embryo demonstrating talin1 expression within the retina/RPE cells lining the CF (arrows) and the hyaloid vasculature (marked by GFP expression from fli1a:eGFP; arrowhead). (G-J) Sagittal sections through the eyes of 48hpf tln1 mutants and siblings stained with Lam-111 (red) and Sytox-green (DNA; green). BM degradation is disrupted in tln1 mutants at 48hpf. (K-N) Maximum projection images of the distal hyaloid in tln1 mutants and siblings demonstrating severe hypotrophy of the hyaloid in the tln1 mutant at 44.5hpf. Scale bar = 20μm (G-J, K,M) and 50μm (L,N).
Figure 6
Figure 6. POM-derived endothelial cells facilitate BM breakdown during CFC
(A-D) Sagittal sections through the eyes of 51hpf clochem378 mutants and siblings stained with Lam-111 (red) and Sytox-green (DNA; green). BM degradation is disrupted in clochem378 mutants in both the (C) proximal and (D) distal regions of the CF when compared to (A,B) siblings. Scale bar = 25um.
Figure 7
Figure 7. Schematics depicting key stages and events of CFC
(A) During Stage I, tissue growth and optic cup morphogenesis generates an appropriate number of cells, which are correctly patterned and positioned in the optic cup. The opposing sides of the CF become closely apposed. During Stage II, the basement membrane lining the CF is degraded through a process involving periocular mesenchyme cells. During Stage III, tissue fusion between opposing sides of the CF closes the fissure. (B) Schematic of basement membrane breakdown (green = BM) within the CF from 31-48hpf. BM breakdown initiates in the central/proximal CF and proceeds bi-directionally, being complete by 48hpf except in the most distal regions of the CF. (C) Schematic of tissue fusion (blue = fusion) in the CF from 41-54hpf. The hyaloid vasculature is depicted in red in each image. Fusion initiates in the central/proximal CF and proceeds bi-directionally, being complete by ~54hpf except in the most distal regions of the CF.
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