Specifications of skeletogenic mesenchyme and chondrocytes in the posterior pharyngeal arches. (A–H) Fluorescence in situ hybridization of barx1 (red) and sox9a (green) at 36 to 120 hpf. Expression domains of barx1 and sox9a mark respective skeletogenic mesenchyme and chondrocytes in pharyngeal arches 3 to 7. (A) At 36 hpf, a single domain expressing barx1 is observed in all pharyngeal arches 3 to 7, with two separate populations (arrows) seen in the dorsal and ventral regions of the second arch. sox9a is partially expressed in a subdomain of the barx1-expressing domain in pharyngal arches 3 to 6 (arrowheads). (B) At 48 hpf, barx1 expression in more anterior pharyngeal arches 3 and 4 gradually reduces, with a single domain of sox9a expression evident in all posterior arches 3–7. (C) At 60hpf, a single domain expressing sox9a is maintained in each posterior arch, with the shape of sox9a expressing domains becoming elongated. barx1 expression domains are seen in the adjacent regions to the sox9a-expressing domains in the posterior arches 3–7. (D) At 72 hpf, the single domain of sox9a expression is further elongated in pharyngeal arches 4-7, with sox9a expression in arch 3 faded. (E) At 84 hpf, sox9a expression is seen in the dorsal regions of arches 4-7, with its expression almost abolished in arch 3. (F) At 96 hpf, sox9a expression is observed in the dorsal regions of arches 4-7, but its expression fades in arch 4. (G) Traces of sox9a expression remain in the dorsal area of arches 4-6 at 108 hpf. (H) At 120 hpf, traces of sox9a expression are seen in the dorsal area of arch 6, with its expression barely seen in the dorsal region of arch 5 and abolished in arch 4. Arrowheads indicate sox9a expression in putative Cbs 3–5. (D–H) From 72 to 120 hpf, barx1 expression is no longer observed in the posterior arches 3–7. (E–H) Expression of barx1 and sox9a in the developing gills is seen in the lower area of (E–H). (A’–H’) Red channel only. (A”–H”) Green channel only. Scale bar: 40 μm. Anterior is to the left. Dorsal is at the top. Cb, ceratobranchial cartilage; D, dorsal; V, ventral. n, number of animals analyzed.
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