Figure 4

Severity of nnt malformations increases with ethanol concentration. (A–L) Untreated and ethanol-exposed Alcian Blue/ Alizarian Red-stained zebrafish embryos at 5 dpf treated from 6 hpf- 24 hpf. All images are ventral views with anterior to the left. (A,B,G,H) Wildtypes and mutants appear phenotypically normal at concentrations of 0 and 0.5% ethanol. (C,I) At 0.75% ethanol, wildtypes still appear normal, but 22.7% of mutants have ceratohyal cartilages that do not extend anteriorly (red arrow). (D,J) At 1% ethanol, the wildtypes remain normal, but 82.2% of nnt mutants had gross craniofacial defects, including a hypoplastic Meckel’s cartilage (yellow arrow). (E,F,K,L).. At 1.25 and 1.5% craniofacial defects become apparent in the wildtypes, and there is increased severity of the craniofacial defects in the mutants. (M) A bar chart depicting total craniofacial malformations across genotype and ethanol concentration (Fisher’s exact test, n ≥ 20 per group, *p  < 0.05, ****p  < 0.0001). (N) A bar chart depicting the types of craniofacial malformations seen in mutants at different ethanol concentrations. EtOH: ethanol, Mut: mutant, Het: heterozygote, Wt: wildtype.