Distinct roles of Fsta and Emx2 in hyoid skeletal development. (A) Dissected mandibular and hyoid skeletons stained with Alcian Blue (cartilage) and Alizarin Red (bone). At 3 dpf, cells between the hyomandibular (Hm) and symplectic (Sy) cartilages and at the forming hyoid joint region stain weakly with Alcian Blue, reflecting their chondrogenic immaturity. By 5 dpf, rearrangements among cells at the Hm-Sy junction result in elongation of Sy, and maturation of cells at the junction fuses Hm and Sy. Cells in the hyoid joint remain immature and weakly Alcian Blue positive. In fsta mutants, the Sy is shortened (red arrowhead), with a build up of chondrocytes in the Hm-Sy junction, and hyoid joint cells inappropriately mature into chondrocytes strongly stained by Alcian Blue, fusing the joint (black arrows). These phenotypes are similar to those of irx7; irx5a mutants. In emx2 mutants, the Hm and Sy cartilages fail to fuse completely (red arrow), and the opercle bone is lost (black arrowhead). emx2; fsta mutants show a compound phenotype, including hyoid joint fusion and reduction of Sy. Hm and Sy are disconnected in emx2; fsta but not emx2; irx7; irx5a mutants. Scale bars: 100 μm. (B) Penetrance of skeletal phenotypes in each genotype: n (sides)=57 (fsta−/−), 42 (emx2−/−), 5 (irx7−/−; irx5a−/−), 11 (emx2−/−; fsta−/−) and 7 (emx2−/−; irx7−/−; irx5a−/−). **P<0.01 versus wild type. (C,D) Two-color in situ hybridization at 36 hpf shows that fsta expression (red) is normal in emx2 mutants, and emx2 expression (red) is normal in fsta mutants. (E) Hyoid joint expression of irx7 at 56 hpf is unaffected in emx2 mutants and reduced in 2/7 fsta; fstb mutants. In green, dlx2a labels arch CNCCs and sox9a labels chondrocytes. Scale bars: 20 μm in C-E. (F) Model for Emx2 and Fsta function in the hyoid arch. At early stages, Fsta promotes irx7 expression, preventing differentiation and cartilage matrix accumulation between Hm and Sy and allowing chondroprogenitors to rearrange into the single stack of Sy chondrocytes. Following rearrangements, Emx2 promotes chondrogenic differentiation to fuse Hm and Sy into a seamless cartilage. By contrast, there is a continuous requirement for Fsta function at the nearby hyoid joint to maintain its patency through active inhibition of chondrogenesis.
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