Figure 4. rasl11b knock down induces a specific curly down phenotype in zebrafish.

(A) Scheme of the rasl11b transcript. The Morpholino targeted sequences are represented by thin red lines. The red boxes correspond to the G1 to G5 domains described in Fig. 1. (B–D) Lateral view of 1 dpf embryos. Both rasl11b MO-ATG and rasl11b MO-GT induce a curly tail down phenotype when injected in WT. This phenotype can be rescued by the coinjection of rasl11b mRNA resistant to MO knock down, rasl11b^MOr (D). (E) In vitro translational assay showing the efficient knock down effect of the rasl11b MO-ATG on wild-type rasl11b mRNA compared to the MO resistant rasl11b^MOr mRNA. (F,G) rasl11b splice-blocking morpholino (rasl11b MO-GT) knocks down endogenous rasl11b mRNA. RT-PCR analysis detected two splice variants that were cloned and sequenced. Sequence comparison revealed that the splice variant (1) resulted from aberrant splicing to an upstream cryptic slice donor site that is present in intron 2, resulting in an early frame shift. The splice variant (2) only missed exon 2 but is not functional (data not shown).