Fig. 2

Requirements of Irx7 and Irx5a in Hyoid Joint Formation

(A and B) Schematics of irx7 and irx5a TALEN mutants show the position of out-of-frame deletions (arrows) relative to homeobox domains (blue boxes).

(C–E) Lateral views of mandibular and hyoid skeletons (left) and magnified views of the hyoid joints (right) show cartilages (Alcian Blue) and bones (Alizarin Red) at 5 dpf. In WT, hyoid joint cells on either side of the Ih cartilage stain poorly with Alcian (black arrowheads). In irx7^el538 and irx7^el538; irx5a^el574 mutants, the hyoid joint is dysmorphic with increased Alcian reactivity (red arrowheads).

(F and G) Dissected adult zebrafish facial skeletons (3-months post-fertilization; size matched). The hyoid joint (dashed box) is magnified in (F′) and (G′) and schematized in (F′′) and (G′′) to show joint fusions in irx7^el538; irx5a^el574 double mutants. Op, opercular bone.

(H) Scheme of neural crest transplants followed by 30–34 hpf heat shocks to induce donor transgene expression.

(I) Whereas control kikGR-expressing cells contributed to hyoid cartilage in 18 of 20 cases, Irx7/GFP-expressing cells contributed to cartilage in only 11 of 72 cases (Fisher′s exact two-tailed test, p < 0.0001).

(J–O) sox10:dsRed hosts received either control hsp70I:Gal4; UAS:kikGR or hsp70I:Gal4; UAS:Irx7-T2A-GFP donor neural crest precursors. Imaging at 2 dpf revealed contribution of donor cells to the mandibular and hyoid arches, and re-imaging at 4 dpf revealed contributions to sox10:dsRed+ hyoid cartilage. Arrowheads indicate contribution of Irx7/GFP-expressing cells to ligaments (white) and bones (magenta).

(P–S) Cell-specific loss of Alcian reactivity is seen in donor cells expressing Irx7/GFP (magnified in Q&prime& S is boxed region from O) but not kikGR (magnified in P′; R is boxed region from K). Numbers indicate proportion of animals showing the displayed phenotype.

Scale bars represent 20 µM unless otherwise noted. See also Figure S2.