Figure 2

(A and B) Translation blocking and splice block morpholinos were used to knock down clvs2 expression in zebrafish. Morpholino efficacy was confirmed through Western blot (A) and reverse transcription PCR (RT-PCR) (B), showing that both morpholinos were able to knock down clvs2 expression. (C) Larval phenotypes were evaluated at 4 days postfertilization (dpf) as having edema (arrows for periorbital and pericardial) or as unaffected (no edema). (D and E) Analysis revealed significantly increased edema phenotypes with clvs2 knockdown compared with controls, and this edema could be rescued by coinjection of wild-type human CLVS1 mRNA but not the p.H310Y variant (E) (*P < 0.05, n > 60 for all groups, 1-way ANOVA). (F) Quantification of excreted GFP-labeled vitamin D in the Tg(lfabp:VDBP-GFP) reporter line revealed a loss of GFP in clvs2 morpholino-injected fish compared with controls (n = 10 for each group, P < 0.0001, 2-tailed t test), demonstrating that GFB integrity was affected by clvs2 knockdown. (G and H) Transmission electron microscopy images show healthy podocyte foot processes with intact slit diaphragms (white arrowheads) around a capillary loop in 5 dpf control morphant larvae and podocyte effacement (red arrowhead) in clvs2 morphants (scale bars = 800 nm and 1 μm, respectively), confirming that edema phenotypes are due to reduced GFB integrity.