Eye morphogenesis in the blind Mexican cavefish

ABSTRACT The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.


Fig. S2. Choosing a candidate gene for transgenesis.
Chosen candidates were Vax1, Vax2, Zic1, Zic2a, Rx3, Lhx2 and Lhx9. (A) Mini-screen of candidate genes by in situ hybridization at different stages (10,12,14,24 and 36hpf) of interest on surface fish and cavefish (not shown) embryos. Anterior is to the left. Dorsal views at 10hpf; lateral views at 14hpf and 36hpf. The eyes were dissected out for Vax1 and Lhx9 (as no eye expression was detected for either of them) to allow better visibility of the inner tissue. Among the 7 genes, 5 were expressed in the anterior neural plate at 10hpf while 2 were not: Vax1 and Vax2, whose expressions were detectable from 12hpf only. Five of them were expressed at least partially in the OV per se (excluding ORR and optic stalk): Vax2, Zic1, Rx3, Lhx9 and Zic2a (faintly). At 36hpf, only 4 of them were still expressed in the optic cup: Zic2a and Zic1 (around the lens), Lhx2 (faintly) and Vax2 (in the ventral retina). Subtle differences between CF and SF expression patterns were observed (not shown), and only one candidate gene was consistently expressed in the eye from neural plate to 36hpf: Zic1.
(B) Dtailed analysis of Zic1 expression pattern at 5 different stages in surface (SF) and cavefish (CF). Anterior is to the left, at 10, 12 and 14hpf, bottom pictures are taken in dorsal view; at 24 and 36hpf, bottom picture are taken in ventral views. Arrowheads indicate an indentation in the eyefield.  Knock-In insertions, based on partial sequencing. Dotted boxes indicate un-sequenced regions, leaving uncertainties. For example, in the surface fish line, there is at least a partial insertion of the repair construct, containing a truncated Hsp70 promoter and at least another insert in the same direction (but potentially several). Of note, the surrounding genomic region is very rich in T and A (GC content around 35%) with many repeats, making PCRs sometimes challenging.
The data show that for both lines the transgenes are inserted at the correct targeted site.  The size of the optic stalk (in a wide meaning: the connection between the OV and the neural tube) is smaller in cavefish during early development due to the smaller size of the OV but rapidly becomes indistinguishable from the optic stalk of the surface fish.   any visible cuts are labelled with a blue asterisk. Additional bands are seen much more frequently and are much more important than with the Cas9 mRNA injection, probably indicating more frequent but also more precocious cut and repair events in the embryo, so that many cells share the same sequence.
(C) Heteroduplex mobility assay: in an electrophoresis, heteroduplexes are slowed down compared to homoduplexes so that they form additional bands that can be seen even if the polymorphism is only a single substitution. In short, the DNA fragments are denatured and renatured to form heteroduplexes. An electrophoresis is then performed (here with a LabChip, PerkinElmer) to detect the presence of polymorphism.
(D) Different cutting and repair events in a single injected embryo. A PCR was performed on one injected embryo (100ng/µL sgRNA2, Cas9mRNA) around the sgRNA2 target site and the product was cloned. Plasmidic preparations from individual colonies were then sequenced.
Various sequences were obtained, evidencing different cut and repair events in one single embryo. sgRNA2 target sequence is highlighted in yellow whenever intact. This F0 fish harboured both insertions and deletions around the cutting site of sgRNA2. A non-injected control fish sequence is included, outlined in black.