Fig. 6: CRISPR mutant snails reveal P. canaliculata pax6 is required for eye formation.

A Schematic representation of the P. canaliculata pax6 genomic region, the gRNA target site, and the crossing strategy used to establish a stable mutant line and to obtain homozygous mutant animals (see Supplementary Fig. 7 and Supplementary Data 5). B Images of wild-type and pax6^−/− 9 dpf embryos and insets of the region at the base of the cephalic tentacle. Wild-type and pax6^+/− animals have eyes while the pax6^−/− snails lack all the eye bulb and eye stalk components. C The percentage of F2 embryos with the “lack of eye” phenotype corresponds to the expected 25%. n = 6 clutches. D Correlation between genotypes and phenotypes in F2 embryos shows that the lack of eyes corresponds to homozygous mutants. n = 3 clutches. E Confocal images of wild-type and pax6^−/− embryos stained with phalloidin during a developmental time course. Phalloidin shows that at 4 dpf there is a first morphological evidence of eye formation through cell wall contraction in the cephalic area of the embryos. The red arrows point at the area where the eye develops (in wild-type and pax6^+/− snails) or where it should be developing (in pax6^−/− snails). F Still images of time lapses showing adult wild-type and pax6^−/− snails free to move in their tanks (see Supplementary Movie 1). Data are represented as mean ± SEM.