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Fig. 3 itch knockdown impairs early zebrafish development.
(A) Splice junction MOs against itcha and itchb efficiently reduced their respective but not opposite mRNAs. RNA was extracted from 48 hpf control embryos or embryos injected with MOs against itcha (MoA) or itchb (MoB). After reverse-transcription, cDNAs from each group were amplified with PCR primers specific for itcha, itchb or GAPDH as a control for 35 cycles. Marked reduction of itcha and itchb was obtained after injection of the appropriate but not the other MO. (B–I) Morphology of embryos injected with MOs targeting itchb (MoB) showed general growth failure, already visible at 24 hpf (B,C) and persisting through to 96 hpf (H,I) as compared to control embryos (CTRL). Scale bar: 500 µm. (J) Total length of control embryos or embryos injected with MOs against itchb (MoB) was measured from the tip of the head to the tip of the tail muscle. Two-factor analysis of variance indicates no significant interaction of injection with age. Pairwise comparison was performed between control and MoB injected embryos at each time point. The number of embryos measured is indicated at the bottom of the graph. ** represents statistical significance at p < 0.01. (K-N) Morphology of control embryos (CTRL), itcha knockdown embryos (MoA), itchb knockdown (MoB) or itcha and itchb knockdown embryos (MoA+B) at 48 hpf. itcha knockdown embryos were slightly delayed, but were morphologically intact as compared to itchb or itcha plus itchb knockdown embryos. (O) Global growth of injected embryos was assessed by total length measurements at 72 hpf. itcha and itchb knockdown embryos were significantly smaller than control embryos, and itchb knockdown were smaller than itcha as assessed by Kruskal-Wallis one-way ANOVA combined with Dunn′s method of group comparison with a significant threshold fixed at p < 0.01 (**). itcha plus itchb knockdown embryos were not significantly smaller than itchb knockdown embryos (n.s.). (P) Growth defect of itchb knockdown was partially rescued with injection of in vitro transcribed human ITCH mRNA. Kruskal-Wallis one-way ANOVA combined with Dunn′s method of comparison established no-significant differences between control embryos (CTRL) and embryos injected with ITCH mRNA alone (mRNA), whereas the total length was significantly reduced in both itchb MO-injected embryos (MoB) and embryos injected with a combination of itchb MO and ITCH mRNA (Rescue). Total length was significantly larger in the Rescue group than in the MoB group, indicating partial rescue. The number of embryos measured is indicated at the bottom of the graphs. Each graph summarizes at least four different experiments.(**) indicates p < 0.01.