Wild‐type zebrafish craniofacial anatomy. Dorsal (A) and ventral (B) views of a 5 dpf whole mount Alcian Blue/Alizarin Red‐stained zebrafish. Our forward genetic screen identified EtOH‐sensitive mutants with qualitative alterations to the craniofacial skeleton. (A) The neurocranium consists of an anterior, neural crest‐derived palatal skeleton, composed of the trabeculae (tr) and ethmoid plate (ep). The palate connects posteriorly to the mesoderm‐derived parachordal cartilages (pc). (B) The viscerocranium consists of 7 segments. The first and second segments have distinct ventral and dorsal skeletal elements at this age. Meckel’s cartilage (mc) and the palatoquadrate (pq) reside ventrally and dorsally, respectively, in the first pharyngeal arch. Ventral and dorsal elements within the second pharyngeal arch are the ceratohyal cartilage (ch) with its associated bone the branchialstegal ray (bsr) and the hyosymplectic cartilage (hs) and opercle bone (op), respectively. The remaining 5 pharyngeal arches house ceratohyal cartilages 1 to 5 (numbered), with the fifth ceratohyal harboring the pharyngeal teeth. The mesoderm‐derived cleithrum (cl) resides just posterior to the pharyngeal arches. Modified from Swartz and colleagues (2014).

The au26 mutation results in susceptibility to EtOH‐induced palatal defects. Flat mounts of 5 dpf EtOH‐treated wild‐type (A) and au26 mutant (B) zebrafish. The anterior neurocranium is severely reduced in EtOH‐treated au26 mutants, and the trabeculae fail to appropriately fuse to the posterior neurocranium (B, arrow).

Lower jaw defects in au15 mutants. EtOH‐treated 5 dpf wild‐type (A) and au15 mutant (B) zebrafish. Meckel’s cartilage (arrowhead), the dentary (d), and the entopterygoid (e) are lost in au15 mutants. The palatoquadrate (arrow) is reduced in au15. In the second pharyngeal arch, the basihyal cartilage (asterisk) is also lost in au15. Ventral views, anterior to left.

Hyomandibular defects in EtOH‐treated au28. The anterior region of the hyomandibular cartilage (A, arrow) is lost in EtOH‐treated au28 mutants (B, arrow). The posterior region of the hyomandibular and the associated opercle bone (op) is not affected. Lateral views, anterior to left.

Bone loss in EtOH‐treated au27 mutants. Flat mounted viscerocrania from wild‐type (A) and au27 mutant (B) zebrafish. There is an overall reduction in the size of craniofacial cartilages in au27 mutants. More striking is the near complete loss of craniofacial bone. Only a tiny remnant of the pharyngeal teeth (arrows) remain in mutants. Op, opercle; cb5, ceratobranchial #5; cl, cleithrum. Anterior to the left.

Bone loss in au27 mutants is not due to a developmental delay. Relative to EtOH‐exposed wild‐type fish (A), ossification in au27 mutants is greatly diminished at 7 dpf. Pm, premaxilla; d, dentary; ra, retroarticular; ch, ceratohyal; op, opercle; bsr, branchiostegal ray. Ventral view, anterior to the left.

Phenotypic classes of au32 mutants. Relative to wild‐type fish (A), the majority of EtOH‐treated au32 individuals have severe reductions to the facial skeleton (B). In a small percentage of au32 individuals, there are gaps in the trabeculae and/or Meckel’s cartilage (C, arrows). Ep, ethmoid plate; tr, trabeculae.

Whole body defects in au32 mutants. Those au32 mutants with more profound craniofacial defects also display marked reductions in the overall body axis (au32, bottom) while less severe au32 mutants (au32, top) have a more normal body axis.

Craniofacial defects in au29 mutants. In untreated au29 mutants (A, B), there are reductions to the ethmoid plate in the neurocranium (A, arrows). Within the viscerocranium, the ceratohyal cartilages (arrow) fail to project anteriorly and the number of ceratobranchial cartilages (numbered) is reduced (B). EtOH exposure dramatically exacerbates these phenotypes (C, D). The ethmoid plate is greatly reduced in EtOH‐treated au29 mutants (C, arrow; the eyes have been dissected away to more clearly visualize the cartilage). (E) Schematic representation of the au29 and au113 alleles. Schematic of exons 14 through 21, of 24 predicted exons, is shown with the location of au29 and au113 noted. The splice donor of exon 15, underlined in wt sequence, is mutated in au29. A 5‐bp indel is present in au113. The PAM sequence is underlined in the wt sequence, and the FspI site used for genotyping is shown in red.

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