Eye morphogenesis driven by epithelial flow into the optic cup facilitated by modulation of bone morphogenetic protein

Abstract

The hemispheric, bi-layered optic cup forms from an oval optic vesicle during early vertebrate eye development through major morphological transformations. The overall basal surface, facing the developing lens, is increasing, while, at the same time, the space basally occupied by individual cells is decreasing. This cannot be explained by the classical view of eye development. Using zebrafish (Danio rerio) as a model, we show that the lens-averted epithelium functions as a reservoir that contributes to the growing neuroretina through epithelial flow around the distal rims of the optic cup. We propose that this flow couples morphogenesis and retinal determination. Our 4D data indicate that future stem cells flow from their origin in the lens-averted domain of the optic vesicle to their destination in the ciliary marginal zone. BMP-mediated inhibition of the flow results in ectopic neuroretina in the RPE domain. Ultimately the ventral fissure fails to close resulting in coloboma.DOI: http://dx.doi.org/10.7554/eLife.05216.001

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…We were able to induce eye-specific loss of pigmentation by expressing our transgene exclusively in the progenitors of the neural retina and the retinal-pigmented epithelium (RPE). For this purpose we used a transgenic line, Tg(rx2:gal4), in which the Gal4 trans-activator is specifically driven in the optic primordium by the promoter of the zebrafish retinal homeobox gene 2 (rx2; Heermann et al 2015). This result confirmed the ability of our strategy to induce Gal4-and Cas9-mediated tissue-specific gene inactivation.…”

Emergence of dorsal-ventral polarity in ESC-derived retinal tissue

“…As seen in vivo, invagination started at the side opposite the fissure, and fissure formation was coupled with the polarized invagination processes. In vertebrate retinal development, it has been thought that the optic fissure is formed by folding of the ventral retinal epithelium (Heermann et al, 2015); however, there are no reports showing a detailed process of optic fissure formation by live imaging. Furthermore, it has previously been shown that increased cell proliferation and extensive apoptosis in the ventral optic vesicle might accompany optic fissure formation (Ozeki et al, 2000;Trousse et al, 2001;Morcillo et al, 2006), and in a Bmp7 mutant mouse, fissure formation was not initiated (Morcillo et al, 2006).…”

2C-Cas9: a versatile tool for clonal analysis of gene function

“…Subsequently, we tested whether spatiotemporally regulated tyr loss-of-function can be induced by our vector system. For this purpose, we used a transgenic line expressing Gal4 under the promoter of the zebrafish retinal homeobox gene 2 (rx2) (Heermann et al 2015), Tg(rx2:Gal4;myl7:GFP). We identified transgenic embryos using the myl7 (myosin, light chain 7, regulatory) promoter, driving heart-specific GFP expression, which was incorporated in the same transgene.…”

“…As a single efficient sgRNA could be identified for this gene, the same target sequence was inserted downstream from each of the two U6 promoters. We chose the rx2:Gal4 driver to express this UAS construct targeting pvalb6 in multipotent retinal progenitor cells as the rx2 promoter is active during early eye field development (Chuang and Raymond 2001;Heermann et al 2015). It has been previously reported that one single RPC can give rise to clones including all of the different retinal cell types spanning all retinal layers (Livesey and Cepko 2001).…”

“…The rx2:Gal4 vector was generated by threeway-Gateway cloning recombining a 5 ′ entry vector carrying the rx2 promoter fragment (Heermann et al 2015) with a Gal4 middle entry, a polyA 3 ′ entry vector, and a myl7:eGFP-Tol2 destination vector (Kwan et al 2007). To generate the rpl5b:Gal4 vector, we cloned a 5700-bp promoter fragment of the ribosomal gene rpl5b in a 5…”