Morpholino-mediated knockdown of aldh6a1 induces dose-dependent developmental defects in zebrafish. (A) Representative bright-field images of zebrafish embryos at 48 and 72 hpf following microinjection with control morpholino (MO-Ctrl), low-dose (0.3 mM) or high-dose (0.5 mM) aldh6a1 splice-blocking morpholino (MO-aldh6a1), and corresponding rescue groups co-injected with aldh6a1 mRNA. (B) Kaplan–Meier survival curves of zebrafish embryos up to 80 hpf. (C) Representative images of individual embryos at 72 hpf illustrating specific malformation phenotypes: 1 tail development failure; 2 tail development failure with pericardial edema; 3 scoliosis; 4 scoliosis with curled tail; 5 scoliosis with curled tail and pericardial edema; 6 scoliosis with curled tail, pericardial edema, and developmental retardation; 7 mild tail curling; 8 delayed yolk sac absorption; 9 delayed yolk sac absorption; 10 mild tail curling with delayed yolk sac absorption. (D) Quantification of normalized hatching rate at 48 hpf. (E) Quantification of total normalized malformation rate at 72 hpf. (F) Dissection of specific malformation types at 72 hpf: spinal curvature (left), tail curling (middle), and pericardial edema (right). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001).
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