Pronephric Grk4 phenotypes are mediated by STAT3. (A) Representative immunofluorescence images of 24 hpf embryos stained for p-Stat3 and total Stat3. Scale bar, 200 μm. (B) Loss of Grk4 increased the percentage of embryos exhibiting stronger p-Stat3 staining than the average control-injected embryo. n=3 experiments with 55-87 embryos in total. **P=0.0022 (data were analyzed using a two-sided Welch's test). (C) In situ hybridization demonstrating nhe3a expression at 48 hpf. Scale bar, 200 μm. (D) Expression of dnSTAT3 rescued the length of nhe3a expression in Grk4-depleted embryos. n=3 experiments with 59-89 embryos in total. ns, P=0.0588; *P=0.0253 and **P=0.0043 (data were analyzed using the Kruskal-Wallis-test with Dunn's multiple comparison test). (E) Embryos expressing dnSTAT3 displayed a weaker nhe3a signal. n=3 experiments with 59-89 embryos in total. *P=0.0147 (data were analyzed usig one-way ANOVA with Sidak's multiple comparison test). (F) dnSTAT3 prevented hydrocephalus development upon Grk4 knockdown. n=4 experiments with 167-188 embryos in total. ****P<0.0001 (data were analyzed using a two-sided Fisher's exact test with the Bonferroni correction applied). Scale bar, 200 μm. (G) Cyst formation was reduced upon dnSTAT3 expression. n=4 experiments with 167-188 embryos in total. ***P=0.0001 and ****P<0.0001 (data were analyzed using a two-sided Fisher's exact test with the Bonferroni correction applied). Scale bar, 100 μm. ns, not significant; Grk4, G protein-coupled receptor kinase 4; STAT3, signal transducer and activator of transcription 3; dnSTAT3, dominant negative STAT3; CTRL, control; MO, morpholino oligonucleotide.
|
