Ethanol decreased col2a1a expression in the ocular neural crest and developing jaw. Treatment with 1% and 3% ethanol from 24 to 48 hpf, followed by wholemount in situ hybridization analysis using a chromogenic (blue) colorimetric assay (Vector Blue Substrate Kit, Vector Laboratories) and FL microscopy analysis of the fluorescent (Far Red/Cy5) colorimetric substrate (Vector Blue Substrate Kit, Vector Laboratories) at 60 and 90 hpf revealed decreased col2a1a expression in the developing eyes and jaws of treated WT Casper zebrafish embryos compared with their untreated and 1% ETOH-treated counterparts. Lateral images (A–C) of wholemount colorimetric in situ analysis at 60 hpf show the teratogenic effects on the ocular development of the treated embryos. The solid and dashed circles highlight the effect on eye size. The black dashed lines indicate the orientation of the plane of section, which passes perpendicular to the spinal column and extends in the rostral-caudal direction. Transverse cephalic sections of the colorimetric in situ analysis (A’–C’) of treated embryos at 60 hpf show a significant decrease in col2a1a expression in the anterior segment [white arrow, iris; blue arrow, hyaloid; orange arrow, sclera, and periocular mesenchyme) of the developing eye with 3% ETOH treatment. The yellow asterisks highlight craniofacial col2a1a expression. Quantification of these effects (E) shows that 3% significantly decreased col2a1a expression. *, p-value ≤ 0.05. Ventral images (F–I) of wholemount colorimetric in situ analysis show significantly delayed jaw development, with severe malformations of the pharyngeal arches (PA) and basihyal (red arrow) and Meckel’s cartilage (white arrow) in treated embryos at 96 hpf. Combined treatment with 3% ETOH from 24–48 hpf and RA from 27–96 hpf increased col2a1a expression in the ocular anterior segment (D’) and partially rescued the teratogenic effects of 3% ethanol treatment on eye development at 60 hpf (D,E) but did not restore col2a1a expression in the craniofacial neural crest (yellow asterisk, (D’)) and further potentiated the deleterious effects of ethanol treatment on the developing jaw (white asterisk, (I)) at 96 hpf.
