Fig. 2

Analysis of ossification of the pharyngeal skeleton of hdac4 zygotic mutants stained using Alcian Blue and Alizarin Red dyes.

(A) Schematic showing elements of the 7 dpf larvae pharyngeal skeleton, lateral view. Cartilage is indicated in blue, bone is indicated in red. (B and C) Total scores assigned to 7 dpf larvae scored for hdac4^aik2 and hdac4^aik3 lines, respectively. (D and E) Representative wild-type larval pharyngeal skeleton (D) and enlarged view (E) of ceratohyal showing small area (less than approximately 20% of total area of element) of bone formation, with even borders, indicated by arrow. Asterisk indicates perichondral bone collar. Lateral view. (F and G) Representative hdac4^aik2 mutant pharyngeal skeleton (F) and enlarged view (G) of ceratohyal showing large area (greater than approximately 20% of total area of element) of bone formation, with irregular borders, indicated by arrow. Asterisks indicate perichondral bone collar. Lateral view. (H and I) Bar graphs comparing ratios of bone to total area of the ceratohyal for hdac4^aik2 and hdac4^aik3 lines. (J and K) Bar graphs comparing ratios of bone to total area of the hyosymplectic for hdac4^aik2 and hdac4^aik3 lines. Bars in graph represent means and error bars are standard errors of means (SEM). H, hdac4^aik2: WT n = 38, mean = 0.102, SEM = 0.013; heterozygote n = 23, mean = 0.113, SEM = 0.017; and mutant n = 32, mean = 0.145, SEM = 0.0112. I, hdac4^aik3: WT n = 17, mean = 0.071, SEM = 0.015; heterozygote n = 28, mean = 0.125, SEM = 0.011; and mutant n = 16 mean = 0.151, SEM = 0.016. J, hdac4^aik2: WT n = 38, mean = 0.097, SEM = 0.009; heterozygote n = 23, mean = 0.109, SEM = 0.015; and mutant n = 32, mean = 0.133, SEM = 0.011. K, hdac4^aik3: WT n = 17, mean = 0.142, SEM = 0.008; heterozygote n = 28, mean = 0.120, SEM = 0.007; and mutant n = 16, mean=0.151, SEM=0.006. Abbreviations: ch, ceratohyal; bsr, branchiostegal ray; de, dentary; en, entopterygoid; hs, hyosymplectic; ih, interhyal; m, Meckel’s cartilage; op, opercle; pq, palatoquadrate; q, quadrate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Scale bar = 100 microns. Cartilage is stained blue (Alcian Blue), bone is stained red (Alizarin Red).