Ectopic expression of USB1 ^P44L in zebrafish. (A) Injections of a higher dose of USB1 mRNA (300 pg/e) were associated with increased mortality of the embryo at 1 dpf (n ≥ 3). Of note, no difference in the mortality between the two doses was registered when injecting the control mRFP1 mRNA. Bars and error bars are averages of the percentage of mortality at 1 dpf and SEM from at least three independent experiments. (B) Tail pigmentation and area of the tail at 2 dpf of zebrafish overexpressing different USB1 variants (n = 4 biological replicates, in orange mean ± SEM). Ordinary one-way ANOVA statistical analysis was performed. ****P < 0.0001. Nonsignificant differences were not annotated. (C) Alcian blue staining at 5 dpf did not reveal any skeletal defects induced by overexpression of the different USB1 variants. Scale bar = 250 μm. (D) Lateral views live-microscopy 5× magnification pictures of tg(mpx:GFP) uninjected and injected embryos with 200 pg/e of the respective construct. Obvious morphological alterations are not reported in uninjected and injected embryos. USB1^P44L-overexpressing embryos presented a decrease in neutrophil count in the tail. Scale bar = 250 μm. (E) Pigmentation normalized to the tail area and tail area of morphants expressing the different USB1 mRNA variants (n = 4 biological replicates, in orange mean ± SEM). Ordinary one-way ANOVA statistical analysis was performed on log₂-transformed data. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Statistically nonsignificant differences were not annotated.