Figure 3. Conditional Yap knockdown in the retina. (A) Principle of reversible and inducible gene knockdown using photo-Morpholinos (photo-MO). Photo-MO contains a photo-sensitive subunit cleaved by 365 nm light. Yap-AS-photo-MO is degraded upon UV light exposure and its translation blocking activity is thus interrupted. Unmodified Yap-MO is rendered inactive by binding to Yap-S-photo-MO. It therefore cannot bind its mRNA target until light-induced cleavage of the sense MO. (B) Diagram of the experimental design. Embryos are microinjected with MO at the two-cell stage, subjected to UV exposure at different developmental stages as indicated (black flashes) and then sacrificed for analyses. (C–F) Analysis of reversible (C, D) and inducible (E, F) Yap knockdown. (C, E) Lateral views and dissected eyes of stage 41 tadpoles following two-cell stage microinjection of the indicated MO (see table in B). (D, F) Quantification of dissected eye area. The stage at which UV exposure was performed is indicated above each bar. The Yap-AS-photo-MO (without any UV exposure) shows the same efficiency as the Yap-MO in reducing eye size. It is efficiently cleaved by UV light since exposure right after injection (stage 4) leads to a wild type phenotype. Restoring Yap function from stage 33/34 or even from stage 37/38 onwards leads to normal eye sized embryos, demonstrating that restricting Yap knockdown to embryogenesis is not sufficient to affect tadpole eye growth. The Yap-S-photo-MO efficiently blocks Yap-MO since their co-injection does not affect eye size. It is efficiently cleaved by UV light since exposure right after co-injection (stage 4) leads to a small eye phenotype, as observed in Yap-MO-injected embryos. Conditional Yap knockdown by light exposure from stage 33/34 or even from stage 37/38 onwards is sufficient to reduce eye size, suggesting that Yap is required at post-embryonic stages to maintain CMZ-dependent eye growth. Of note, in our experimental conditions, UV light exposure does not generate any significant effects on eye size (data not shown). The number of analyzed tadpoles is indicated within each bar. Scale bar = 1 mm. DOI: http://dx.doi.org/10.7554/eLife.08488.009
Image published in: Cabochette P et al. (2015)
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