Histological analysis of dlx mutants using alcian blue and alizarin red showing altered lengths of MC, PQ and CH cartilage structures at 5 dpf. (A–G) Histology stains were performed in at least 15 larvae for each genotype and presented in the ventral (A–G) or lateral (A′–G′) Scale = 150 μm. (H–K) Flat mount images in ventral position of dlx5a−/− (n = 5) and dlx5i6−/− (n = 5). Scale = 100 μm. (L) Schematic showing how structures were measured. Lengths of both halves of MC (red), PQ (yellow) and CH (orange) were measured, then averaged for each animal. Averages were then calculated per genotype. The length (l) and width (w) of the MC were also measured. Dashed line represents entire head length. (M–O) Average lengths of MC (M), PQ (N) and CH (O) in WT (n = 33), dlx1a−/− (n = 12), dlx2a−/− (n = 14), dlx5a−/− (n = 8), dlx5i6−/− (n = 13), dlx6a−/− (n = 17) and inter56−/− (n = 16) larvae. Results are reported as mean ± SEM. (P) l/w ratio in dlx5a−/− and dlx5i6−/− larvae were smaller compared to WT. One-way ANOVA with Tukey’s multiple comparisons test was performed. * p < 0.05; ** p < 0.001; *** p < 0.0001.
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