Generation of a zebrafish nnt mutation. (A) Schematic depicting the 74 bp deletion in exon 4 of nnt. The gRNA binding site (gRNA jke888) and PAM sequence are noted. (B) Location of the mutation (designated au111) relative to the 22 exon long gene. (C) Confocal images of fluorescent in situ hybridization staining for nnt-specific probe in 24 hpf wildtype and (D) mutant embryos, demonstrating a lack of transcript in nnt mutants, suggesting extensive non-sense mediated decay of the mutant transcript. Anterior is left, dorsal is up, e: eye, ea: ear.

Loss of nnt sensitizes embryos to ethanol teratogenesis. (A–D) Alcian Blue/Alizarian Red-stained zebrafish at 5 dpf treated from 6 hpf- 5 dpf. All images are ventral views with anterior to the left. (A,B) Control and ethanol-treated wildtypes and (C,D)nnt mutants. Both the untreated and 1% ethanol dosed wt embryos appear phenotypically normal. (C) Untreated nnt mutant with a typical phenotype akin to wt. (D) Ethanol-exposed nnt mutant zebrafish with aberrant craniofacial phenotype consisting of a hypoplastic Meckel’s cartilage (yellow arrow), deformed ceratohyal (red arrow), microphthalmia, and microcephaly. (E) Quantification of the percentage of fish with craniofacial defects in each group (Fischer’s exact test, n ≥ 20 per group, ****p < .0001). Only ethanol-treated mutants displayed craniofacial defects, a statistically significant increase. EtOH: ethanol, Ctrl: control, mut: mutant, het: heterozygote, Wt: wildtype.

nnt mutants appear sensitized to ethanol teratogenesis from 6 to 24 hpf. Embryos were treated with 1% ethanol at different timepoints (A–H) to establish a critical period of ethanol teratogenesis. All images are ventral views with anterior to the left. Control (unexposed) wildtype and mutant (A, E) appear phenotypically normal. Wildtype embryos dosed across all timepoints (B–D) appear phenotypically normal. (F) Mutant embryos dosed at 6–24 hpf had stark craniofacial defects consisting of a hypoplastic Meckel’s cartilage (yellow arrow), deformed ceratohyal (red arrow), microphthalmia, and microcephaly. (G) Mutant embryos dosed at 24–48 hpf had no apparent defects in craniofacial morphology, but did have microcephaly and reductions in the size of skeletal elements. (H) Mutant embryos dosed at 48–72 hpf had no apparent defects. (I) Quantification of the percentage of fish with craniofacial defects in each group. (Fischer’s exact test, n ≥ 20 per group, **p  < 0.01, ****p  < .0001). The most highly statistical difference was observed at 6–24 hpf. Mut: mutant, Het: heterozygote, Wt: wildtype.

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.

ROS concentration is elevated in nnt mutants exposed to ethanol. (A–H) CellROX Assay-stained 24 hpf fish that were unexposed, exposed to 1% EtOH from 6–24 hpf, exposed to NAC from 6–24 hpf, or exposed to 1% EtOH +1 mM NAC from 6–24 hpf. All images are dorsal views of the head with anterior to the left. (A) Unexposed wildtype embryos have lower basal levels of ROS than (E) unexposed mutants (p = 0.0175). (B) 1% EtOH exposed wildtypes have lower ROS levels than (F) EtOH-exposed mutants (p = 0.0022). (C) 1 mM NAC treated wildtypes were not significantly different from (G) NAC treated mutants. (D) Wildtypes treated with 1 mM NAC and 1% EtOH were not significantly different from the (H) NAC and EtOH-treated mutant, but these mutants had significantly elevated ROS compared to the wildtype control (p = 0.0016). (I) Graph depicting ROS concentration across all groups in 24 hpf zebrafish (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (J–Q) CellROX Assay-stained 48 hpf fish. Dorsal views, anterior to the left. (J) Unexposed wildtypes were not significantly different in ROS concentration than (N) unexposed mutants. (K) 1% EtOH-exposed wildtypes were not significantly different from (O) EtOH-exposed mutants. (L) Wildtypes treated with 1 mM NAC were not significantly different from (P) NAC-treated mutants. (M) Wildtypes treated with 1 mM NAC and 1% EtOH were not significantly different from the (Q) NAC and EtOH-treated mutant, though these mutants had significantly lower ROS concentration compared to the EtOH-treated mutants (p = 0.0337). (R) Graph depicting ROS concentration across all groups in 48 hpf zebrafish (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Ctrl: control, EtOH: ethanol, NAC: N-acetyl Cysteine, Mut: mutant, Wt: wildtype.

Aberrant ethanol-induced apoptosis in nnt mutants is rescued by antioxidant treatment. (A–F) 36 hpf nnt;fli1a:eGFP (green) embryos stained with TUNEL (red) (n = 14 per group). Lateral views, anterior to the left, dorsal up. (A) Unexposed wildtypes had significantly lower apoptosis in the pharyngeal arches compared to (D) unexposed mutants (p = 0.0230). (B) 1% ethanol-dosed wildtypes had significantly less apoptosis in the arches and brain compared to (E) ethanol-treated mutants (p = <0.0001 and p = 0.0003). (C) 1 mM NAC + 1% EtOH dosed wildtypes were not significantly different from (F) NAC + EtOH-treated mutants (p = 0.7775), but NAC + EtOH mutants had significantly lower apoptosis in the arches and brain compared to ethanol-dosed mutants (p = <0001 and p = 0.0491, respectively). (G–J) Schematic of the spatial distribution of apoptosis in the brains (blue arrow) and pharyngeal arches (red arrow) of untreated and ethanol-treated mutants and wildtypes (n = 5 per group). (G) The wildtype control had fewer apoptotic cells that were more widely distributed across the brain and arches compared to the (H,I) unexposed mutants and ethanol-treated wildtypes that had higher levels of apoptosis in both regions. (J) Ethanol-treated mutants had more apoptosis than all other groups and had cell death localized to the ventral portion of the arches and clustered around the midbrain-hindbrain boundary. (K) Graph depicting apoptotic cells in the neural crest across all groups (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 14 per group, *p < 0.05, **p < 0.01, ****p < 0.0001). (L) Graph depicting apoptotic cells in the brain across all groups (Two-way ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 14 per group, *p < 0.05, **p < 0.01, ***p < 0.001). Ctrl: control, EtOH: ethanol, NAC: N-acetyl Cysteine, Mut: mutant, Wt: wildtype.

Proliferation is not significantly altered in neural crest of nnt mutants. (A–D) 36 hpf nnt;fli1a:eGFP (green) embryos stained with pHH3 (red). Lateral views, anterior to the left, dorsal up. (A) Unexposed wildtypes and (C) mutants have similar levels of proliferating cells compared to (B) ethanol-treated wildtypes and (D) mutants. (E) Graph depicting proliferating cells in the neural crest (Twoway ANOVA with multiple comparisons, black bars depict mean ± SEM, n = 14 per group). Ctrl: control, EtOH: ethanol, Mut: mutant, Wt: wildtype.

Ethanol-induced craniofacial abnormalities are rescued by concurrent NAC dosage. (A,B) Flatmounts of 5 dpf wildtype craniofacial skeleton displaying the linear measurements used with anterior to the left. (A) Viscerocranium and (B) neurocranium. The cartilaginous elements that were measured: m: Meckel’s length, cl: ceratohyal length, tl: Trabeculae length, tw: Intertrabecular width, el: Ethmoid plate length, ew: Ethmoid plate width, an: Anterior neurocranium length, pn: Posterior neurocranium length. (C–J) Embryo were unexposed, exposed to 1% EtOH from 6–24 hpf, or exposed to 1% EtOH +1 mM NAC from 6–24 hpf. Fish were then grown to 5 dpf for morphometric analyses. Graphs depict craniofacial measurements across each cartilaginous element in all groups (Two-way ANOVA with multiple comparisons, black bars depict range, n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). In nearly every instance in which ethanol caused a significant reduction in length or width in nnt mutants, the co-exposure with NAC restored the size back to that of wildtype (C,D,G-I). The only exception being posterior neurocranium length, where the co-exposure is not significantly different from wildtype or ethanol-exposed nnt mutants. Ctrl: control, EtOH: ethanol, NAC: N-acetyl Cysteine, Mut: mutant, Wt: wildtype.