Fig. 4.

The pronephric phenotypes of stk3^−/− mutants involve activation of Wwtr1. (A) Immunofluorescent staining of Wwtr1, PKCζ and DAPI in cryostat sections of stk3^−/− larvae and WT siblings at 12 dpf. Merged images are presented with Wwtr1 staining in green, PKCζ in red and DAPI in blue. Dashed line loops indicate the renal tubules. The arrowheads indicate the nuclear accumulation of Wwtr1. Scale bar: 20 μm. (B) Morphology of larvae at 21 dpf. As no stk3^−/−;wwtr1^+/+ fish could be found at 21 dpf, we presented stk3^−/−;wwtr1^+/+ fish at 15 dpf for comparison. Asterisks indicate the edema. Scale bar: 1 mm. (C) Kaplan–Meier plot for the onset of edema in progeny of stk3^+/−;wwtr1^+/− in-cross. stk3^−/−;wwtr1^−/− double mutants exhibited delayed edema onset compared to stk3^−/− single mutants. Statistical analysis was performed using log-rank (Mantel–Cox) test (P<0.001). (D) H&E staining of paraffin sections of larvae at 21 dpf. stk3^−/−;wwtr1^−/− double mutants exhibited mild glomerular cyst with enlarged Bowman's space (asterisks) and pronephric tubule (arrowheads) dilation. As no stk3^−/−;wwtr1^+/+ fish could be found at 21 dpf, we presented stk3^−/−;wwtr1^+/+ fish at 15 dpf for comparison. Scale bar: 50 μm. (E) Statistical analysis of relative renal corpuscle areas [renal corpuscle area/(trunk width)²]. stk3^−/−;wwtr1^−/− double mutants exhibited significantly smaller renal corpuscles than stk3^−/− single mutants (stk3^−/−;wwtr1^+/+). Different letters indicate statistical significance by one-way ANOVA, mean±s.e.m. [n=10 stk3^+/+;wwtr1^+/+ (n=5 at 15 dpf, n=5 at 21 dpf), n=7 stk3^−/−;wwtr1^+/+ (15 dpf), n=6 stk3^−/−;wwtr1^−/− (21 dpf); as the edema onset times were different in stk3^−/−;wwtr1^+/+ and stk3^−/−;wwtr1^−/− lines, we sampled the fish at their median edema onset times]. a.u., arbitrary units.