Table I. Ethmoid plate cartilage width (in micrometers) at 4.5 dpf in ethanol and retinoic acid treated embryos.
Embryos were treated from 3 hpf until 24 hpf (during gastrulation and somitogenesis) with 100 mM ethanol, 10−9 M retinoic acid, or 100 mM ethanol and 10−9 M retinoic acid together, and compared to untreated control embryos. At 4.5 dpf larvae were fixed using paraformaldehyde to stop development, and processed for alcian blue staining to visualize developing craniofacial cartilages (see Materials and Methods). Differential interference contrast (DIC) microscopy images focused on the dorsal craniofacial cartilage structures were used to measure the width of the ethmoid plate using ImageJ image analysis software (see Materials and Methods). Two-way ANOVA was performed to compare treatment groups. Ethanol-by-retinoic acid interaction was significant (P=0.0703), and pairwise comparison P-values are shown. Bonferroni adjustment for multiple (six) comparisons was used to ensure an overall ninety five percent confidence. Thus, P-value≤0.008 was used to conclude that groups were statistically different.
| Control | Ethanol | Ethanol+RA | RA only | |
|---|---|---|---|---|
| Mean | 148.4 | 134.1 | 154.8 | 158.1 |
| SD | 6.2 | 10.4 | 6.5 | 5.5 |
| n | 7 | 9 | 6 | 7 |
| P (vs Control) | 0.0012 | 0.1503 | 0.0274 | |
| P (vs Ethanol) | <0.0001 | <0.0001 | ||
| P (vs RA) | 0.4503 |