Fig. 4

The bone progenitor markers runx2a and runx2b are differentially expressed in kat2a^−/− and kat2b^−/− zebrafish embryos and Kat2a^hat/hat mouse embryos. In situ hybridization for runx2a (A–C) and runx2b (D–G) in WT or heterozygous (A,D,E), kat2a^−/− (B,F,G), and (C) kat2b^−/− 72 hpf zebrafish embryos. (A–D,F) ventral views, (E,G) lateral views, with the anterior to the left in all. kat2a^−/− (B) and kat2b^−/− (C) show reduced expression of runx2a, especially in the anterior maxilla, whereas expression in the dentary bone remains strong as compared to controls (A). runx2b expression in forming bone elements is reduced overall in kat2a mutants (F,G) compared to the control (D,E). For runx2a, n = 9/34 for kat2a, n = 8/31 for kat2b. For runx2b, n = 8/30 for kat2a, n = 8/29 for kat2b. (H–L) Whole-mount in situ hybridization with Runx2 and Bmp4 antisense probes on mouse E12.5 Kat2a^Wt/hat (H,J) and Kat2a^hat/hat (I,K) embryos. (L) RT-qPCR quantification of Runx2 and Bmp4 expression with RNA extracted from E12.5 facial prominences of control (Kat2a^Wt/hat; Ctrl on graph) and mutant (Kat2a^hat/hat; Mut on graph) embryos. The yellow outline in (H) marks the area dissected for RNA extraction. Gene 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. Neural tube closure defects are evident in Kat2a^hat/hat mutants. bs, branchiostegal ray; cb-5, ceratobranchial-5; ch, ceratohyal; cl, cleithrum; de, dentary; ep, ethmoid plate; hs, hyosymplectic; md, mandible; mx, maxilla; op, opercle. The scale bars represent 100 μm.