Fig. 6

Effect of knockdown of the PI3K ortholog on ISVs and the rescue effects of wild-type wnk1a, wnk1a containing an Akt phosphorylation site mutation and kinase-deficient wnk1a on flk1 morphants.

(A) The effects of pi3kc2α MO on the length of intersegmental vessels. (B) Two Thr35 mutations and one Lys206 mutation were generated using site-directed mutagenesis. The sequence of wild-type wnk1a aligned with Thr35 and Lys206 mutants, which are Akt phosphorylation site and kinase domain mutants, respectively. (C) Co-injection of wnk1a mRNA rescues flk1 morphants. Quantitative analysis of the length of the ISVs in flk1 morphants co-injected with various wnk1a mRNAs. Experiments were performed at least three times, and the number of embryos analyzed is shown in the bar graph. Red indicates that the ISVs grew to full length, orange indicates that the ISVs were 75% of the normal length, yellow indicates 50%, green indicates 25%, and light blue indicates no ISVs were observed in the embryos. The differences between treatments were assessed using a two-tailed Student’s t-test. Significant differences between the morphants and controls are indicated (_*, P<0.05; _**, P<0.01; and _***, P<0.001).