Fig. 3

Cartilage progenitor expression of sox9 and col2a1 is reduced in kat2a^−/− and kat2b^−/− zebrafish embryos and E12.5 Kat2a^hat/hat mice. Whole-mount in situ hybridization for zebrafish sox9a (A–L) and col2a1 (M–O) in wildtype or heterozygous embryos (A–D,M), kat2a^−/− (E–H,N), and kat2b^−/− (I–L,O) zebrafish embryos at 48 hpf (A,B,E,F,I,J) and 72 hpf (C,D,G,H,K–O). Lateral views (A,C,E,G,I,K), and ventral views (B,D,F,H,J,L–O), with the anterior to the left in all cases. At both stages, sox9a and col2a1 expression is reduced in the forming cartilage elements. At 48 hpf, there is an overall reduction, especially in the anterior expression domain of sox9a (E, F and I, J as compared to control (A,B)). At 72 hpf, the number of cartilage elements is reduced, especially in the ceratobranchial cartilages in both kat2a (G,H,N) and kat2b (K,L,O) as detected with both sox9a and col2al. For sox9, n = 8/33 for kat2a, n = 7/30 for kat2b. For col2a1, n = 7/29 for kat2a, n = 7/31 for kat2b. (P–S) Sectional in situ hybridization with a Sox9 antisense probe on mouse E12.5 Kat2a^Wt/hat (P–R) and Kat2a^hat/hat (Q–S) cryosections through the frontonasal prominence (P,Q) and the mandibular prominence (R,S) as schematized in the diagram of a E12.5 embryo adapted from the eHistology Atlas (T, the red bar indicates the plane of transverse sections). M = Meckel’s cartilage, T = Tongue. (U) RT-qPCR quantification of Sox9 and Col2a1 expression with RNA extracted from the facial prominences of control (Kat2a^Wt/hat; CTRL on graph) and mutant (Kat2a^hat/hat; MUT on graph) E12.5 mouse embryos. Expression was normalized to GusB, n = 4. The Y-axis shows the Log2 fold change of the relative gene expression, following the ddCT method; * denotes statistical significance (p < 0.05) using Welch’s t-Test. The error bars show the standard error of the mean. cbs1-5, ceratobranchials 1-5; ch, ceratohyal; ep, ethmoid plate; m, Meckel’s cartilage; n, neurocranium; pas, pharyngeal arches. The scale bars represent 100 μm.