Figure 6. Impaired eye gastrulation results in coloboma.

(A–B) Membrane-localized GFP (rx2::GFPcaax) in a developing eye during optic fissure closure (A = early, B = late) (lateral view, derived from imaging data, (A) start at 24 hpf, (B) after 34 hr imaging at 22°C). Rx2 is expressed in retinal stem cells/RPCs (A) and after NR differentiation is additionally expressed in photoreceptors and Müller Glia (B) while its expression is maintained in retinal stem cells of the CMZ (Reinhardt and Centanin et al., submitted). The optic fissure margins are still undifferentiated (arrows in B), (C) developing eye of rx2::BMP4 fish (lateral view), membrane-localized GFP (rx2::GFPcaax, anti-GFP immunointensified), DAPI nuclear labeling and anti-laminin immunostaining, the optic fissure is visible, noteworthy the temporal retina is mis-shaped and folded into the RPE domain (best visible in DAPI, arrowheads), and located on a basal membrane (arrowheads in anti-laminin), especially the temporal optic fissure margin (arrowheads in GFPcaax) is located in the folded part of the temporal retina and not facing the optic fissure (arrows in GFPcaax) (24 hpf) (D–E) impaired optic fissure closure in rx2::BMP4 embryos over time at a proximal (E) and a distal (D) level. (Data obtained from 4D imaging of rx2 ::BMP4/ rx2::GFPcaax started at 21.5 hr. Data are also presented as Video 10.) Importantly, next to the affected temporal optic cup also the nasal optic cup is mis-folded (arrowheads in D). Remarkably, however, the nasal optic fissure margin extents into the optic fissure (dashed arrow in E) but the temporal optic fissure margin does not, likely being the result of the intense mis-bending of the temporal optic cup. This results in a remaining optic fissure (asterisk in E). (F–G) Brightfield images of variable phenotype intensities observed in rx2::BMP4 hatchlings.

DOI: http://dx.doi.org/10.7554/eLife.05216.016

Figure 6—figure supplement 1. Postembryonic eye development of rx2::BMP4 hatchlings.

Although the temporal optic cup is largely malformed and folded it can be seen clearly, that vsx1 as well as vsx2 transgenes (intensified by wholemount immunohistochemistry) are expressed in the folded epithelium (arrows). This indicates at least a partial correct differentiation into neuroretinal tissue.

DOI: http://dx.doi.org/10.7554/eLife.05216.017

Figure 6—figure supplement 2. Lateral view on optic cup development over time, rx2::GFPcaax (control) is compared to rx2::BMP4 at proximal and distal levels.

While in controls the lens-averted domain (yellow dotted line) is integrated into the developing optic cup it persists in rx2::BMP4 embryos. Note the increasing optic fissure (arrows) in rx2::BMP4 embryos. These data were obtained by 4D imaging (start at 20 hpf) and are also presented as Video 8 and Video 9.

DOI: http://dx.doi.org/10.7554/eLife.05216.018

Figure 6—figure supplement 3. Dorsal view on optic cup development of an rx2::BMP4 embryo over time at ventral vs central/ dorsal levels.

The yellow dotted line indicates the border between the lens-facing and the lens-averted domain. Remarkably, the lens-averted domain is not integrated into the optic cup (compare to Figures 1 and 2). Notably, an altered morphology of the ventral optic vesicle can be observed showing the domain which is not going to be integrated (arrows). These data are derived from imaging data (start at 19 hpf) which is presented in Video 7.

DOI: http://dx.doi.org/10.7554/eLife.05216.019

Figure 6—figure supplement 4. Ventral retinal identity remains in rx2::BMP4 embryos.

Whole mount in situ hybridization with a vax2 probe (NBT/BCIP) of control (left) and rx2::BMP4 embryos (right) in a lateral view (upper pictures) and a ventral view (lower pictures) (28 hpf). Note that the ventral retinal marker remains expressed in the forming ventral optic cup even if BMP4 is expressed panocularly (rx2::BMP4). Also note that the vax2 domain in control is broader than in the rx2::BMP4 embryos (arrows) in which it is more prominent in the optic stalk region (arrowhead) (the dotted line indicates the optic fissure).

DOI: http://dx.doi.org/10.7554/eLife.05216.020