Fig. 8. Sox9-depleted embryos develop abnormal pharyngeal arches and altered pattern of skeletal elements. (A) Ventral view of a tailbud stage embryo injected with 5 ng of Sox9-AS and RNA encoding the lineage tracer ß-galactosidase in one blastomere at the eight-cell stage. The bracket indicates the pharyngeal arches on the uninjected side. On the injected side (arrow, red ß-galactosidase staining), the pharyngeal arches are missing. (B) Longitudinal section of an embryo similar to the one presented in A. Note the absence of well-defined pharyngeal arches on the injected side (arrow) when compared with the control side (bracket). The asterisks indicate individual pharyngeal arch. cg, cement gland. Migration (C) and contribution to cranial skeletal elements (D) of individual neural crest segments. Modified from Sadaghiani and Thiebaud (Sadaghiani and Thiebaud, 1987). Meckel’s (Me), quadrate (Qu), ethmoid (Et), cerathoyal (Ce), basihyal (Ba) and branchial/gills (Br) cartilages. Embryos were co-injected in one blastomere on the dorsal side at the eight-cell stage with 3 ng of Co-AS (E,F) or 3 ng of Sox9-AS (G,H) and RNA encoding the lineage tracer ß-galactosidase. At stage 45, tadpoles were fixed and stained for ß-galactosidase to identify the injected side (red staining), right side in all cases. Dorsal (E,G) and ventral (F,H) views indicate an overall reduction of the cranial structures in Sox9-AS-injected embryos (G,H). (I-K) Flat-mounts of Alcian Blue stained skeletal structures from Stage 45 tadpoles. Injected side is on the left. (I) Normal pattern of skeletal elements in a Co-AS-injected embryo. (J,K) Sox9-AS-injected embryos presenting different levels of skeletal defects including loss of Meckel’s cartilage and reduction of ceratohyal and branchial cartilages. Color-coded arrows indicate the origin of each skeletal element according to D. The mesoderm-derived basihyal (black arrows) and infrarostral (black arrowheads) cartilages are unaffected in Sox9-AS-injected embryo (J,K).
