Cell Behaviors during Closure of the Choroid Fissure in the Developing Eye

Abstract

Coloboma is a defect in the morphogenesis of the eye that is a consequence of failure of choroid fissure fusion. It is among the most common congenital defects in humans and can significantly impact vision. However, very little is known about the cellular mechanisms that regulate choroid fissure closure. Using high-resolution confocal imaging of the zebrafish optic cup, we find that apico-basal polarity is re-modeled in cells lining the fissure in proximal to distal and inner to outer gradients during fusion. This process is accompanied by cell proliferation, displacement of vasculature, and contact between cells lining the choroid fissure and periocular mesenchyme (POM). To investigate the role of POM cells in closure of the fissure, we transplanted optic vesicles onto the yolk, allowing them to develop in a situation where they are depleted of POM. The choroid fissure forms normally in ectopic eyes but fusion fails in this condition, despite timely apposition of the nasal and temporal lips of the retina. This study resolves some of the cell behaviors underlying choroid fissure fusion and supports a role for POM in choroid fissure fusion.

Keywords: choroid fissure; coloboma; eye; morphogenesis; optic fissure; optic vesicle; periocular mesenchyme; zebrafish.

Figure 1

Cell organization and behavior during choroid fissure fusion. (A) Schematic showing the optic cup in parasagittal section at the level of the lens just prior to choroid fissure fusion. The cells lining the fissure margins are continuous with the retinal pigmented epithelium and approach each other with their basal surfaces (green) facing the fissure and their apical surfaces (magenta) at the sub-retinal space (ventricular lumen of the optic vesicle). Boxed region corresponds to retinal regions shown in (B–I). Dorsal is up. (A') Schematic view of the optic cup midway through choroid fissure fusion. Letters (B–E) correspond to the positions of schematics/sections in (B–E, B'–E'). (B–E) Sections through the choroid fissure during fusion showing apically-located zonula occludens labeling (ZO1; magenta) and the basal lamina component Laminin (green) in the ventral retina of a 44 hpf embryo. (B'–E') Schematic of progressive steps in choroid fissure fusion process based on analysis of eyes from 8 embryos at 44 hpf (as in B–E). (F–L) Various views of the closing choroid fissure with transgenic lines analyzed or protein derived from injected RNA indicated to the left of the panels. (F) Image stills over a 4 h period from a 10 h time-lapse movie (Movie S1) from 34 to 44 hpf of Tg(ctnna:citrine)^ct3aGT labeling of α-catenin-labeled adherens junctions in the fusing choroid fissure. (G) Z-stack images at two depths of the choroid fissure showing cell shapes just prior to and at the point of fusion in a 44 hpf eye. (H) Image stills over 11.5 h from a time lapse movie (Movie S3) from 34 to 45.5 hpf showing transplanted cells expressing mRFP (magenta) in an eye in which all cells are expressing nuclear H2B-GFP (gray in t = 0); one cell located in the fissure has been pseudocolored in green and the H2B-GFP fluorescence removed from the four panels on the right. Green dashes highlight the position of the fissure (visible when viewing H2B-GFP expression). (I) Image stills over 6 h 40 m from a 12 h time lapse movie (Movie S4) from 34 to 46 hpf showing transplanted cells in the fissure expressing cytoplasmic-GFP in an eye expressing mRFP. The fissure cell divides once during the course of the movie. (J) Image stills over 11 h from a 12 h time lapse movie (Movie S5) from 26 to 38 hpf showing POM cells expressing CAAX-GFP (pseudocolored in green). (K) Image of the choroid fissure prior to fusion showing apically-located GFP-tagged Pard3 labeling (green) and nuclear staining (gray) in the ventral retina of a 40 hpf embryo. The image shows that cells lining the choroid fissure (CF) are in continuity with the retinal pigment epithelium (RPE) and the pseudo-stratified/stratified neural retina (NR). (L,M) Image stills over 6.5 h from a 12 h time lapse movie (Movie S6) from 34 to 46 hpf showing the superficial displacement of the hyaloid vessel during choroid fissure closure. The orientation in K is as other panels above; in L, the image has been rotated to give a lateral view into the fissure; the irregular spots inside the white dashed lines (outlining the hyaloid vessel) are a result of movement of blood cells. The superficial displacement of the vessel is shown by the red arrow. Scale bars: (B–I) ~10 μM; (J,K) ~30 μM.

Figure 2

Cell death is not required for choroid fissure fusion. Images of whole eyes (A–B') or Z-stacks of the retina (C–C') of wildtype (left column) and NO067 mutant (right column) embryos at ages shown bottom right. Arrows show position of the choroid fissure. (A–B') TUNEL labeling (blue) of dying cells. There are more apoptotic cells in the mutant eye but they do not localize to the choroid fissure. (C–C') DAPI staining of nuclei showing the retina of the mutant eye has undergone normal morphogenesis in the absence of macrophages. Scale bar: 50 μM.

Figure 3

Failure of choroid fissure fusion in eyes derived from optic vesicles with reduced POM. (A–C) Brightfield images of two host embryos at different stages after optic vesicle transplantation (at 12 ss, about 14 h pf), showing location of the ectopic eye on the yolk. Arrowheads indicate the open choroid fissure in the ectopic eyes. (D,E) Methylene blue stained sagittal sections at low (D) and high (E) resolution through an ectopic eye at 5 dpf. (F–O) Images of control (F–J) and ectopic (K–O) eyes imaged either in brightfield (F,K,I,N) or by confocal microscopy (G,H,L,M,J,O) showing cell membranes (mRFP, gray) and sox10:GFP labeled POM (green) at ages shown on the panels. Arrowheads indicate the choroid fissure. Asterisk in O' indicates retinal ganglion cell axons. The position of the lens is indicated with a white circle in (H) and (M). ee, ectopic eye; i, intestine; sc, spinal cord; y, yolk. Scale bars 50 μM.