FIGURE 3

FIGURE 3

Budding of epibranchial cartilage 4 at the dorsal end of ceratobranchial cartilage 4. (A–F) Alcian Blue (cartilage) and Alizarin Red (bone) staining visualizing Cbs. (A) Arrowhead indicates rectangular and mono-layered Alcian Blue-positive cartilage cells in the dorsal end of Cb4. 8 out of 10 animals show mono-layered cartilage cells, whereas 2 out of 10 animals show multi-layered cartilage cells presented in (B). (B) A population of round and multi-layered cartilage cells in the dorsal end of Cb4 is marked with an arrowhead. 7 out of 12 animals show multi-layered cartilage cells, whereas 5 out of 12 animals show mono-layered cartilage cells. (C) Arrowhead indicates the extended population of round and multi-layered cartilage cells to the dorsal end of Cb4. 16 out of 23 animals show multi-layered cartilage cells, whereas 2 out of 23 animals show mono-layered cartilage cells. The remaining 5 out of the 23 animals form the Eb4 bud presented in (D). (D) Arrow marks the Eb4 budding anterior-dorsally at the dorsal end of Cb4, with a red arrow marking the posterior dorsal tip. 5 out of 23 animals display the Eb4 bud, with the remaining 18 out of the 23 animals showing mono- or multi-layered cartilage cells but no Eb4 bud, as presented in (A–C). (E) Arrow indicates the Eb4 bud, with increasing cartilage cells in the Eb4 bud, at the dorsal end of Cb4. Red arrow marks the posterior dorsal tip of Cb4. 8 out of 13 animals show an increase in the Eb4 bud, whereas 1 out of 13 animals shows multi-layered cartilage cells, yet no Eb4 bud formation. The remaining 4 out of the 13 animals show a growing Eb4 presented in (F). (F) The growing Eb4 and the evident AECb4 at the dorsal end of Cb4 are marked with black and red arrows, respectively. (G–L) Cartoons of Cb4 forming Eb4 drawn based on the corresponding (A–F). (M–R) Confocal images of Cbs in Tg(sox10:GFP) transgenic animals during Eb formation. (M)sox10-positive cells label the posterior pharyngeal arches (A3 to A7) in 3.0 mm SL zebrafish. (N) Arrowheads indicate the dorsal ends of Cbs 1-5 in 3.5 mm SL zebrafish. Before Eb4 forms at the dorsal end of Cb4, no distinct populations of sox10-positive cells are observed around the dorsal regions of Cbs 1–5. (O–R) Yellow and white arrows mark Ebs, with arrowheads indicating the dorsal ends of corresponding Cbs in 4.0 (O) 4.5 (P) 5.0 (Q) and 5.5 mm SL zebrafish (R). No separate populations of sox10-positive cells are seen around the dorsal regions of respective Cbs before Eb formation. AECb4 is marked with red arrows. Scale bar: 40 μm. Anterior is to the left. Dorsal is at the top. A, arch; Cb, ceratobranchial cartilage; Eb, epibranchial cartilage; AECb4, accessory element of the ceratobranchial 4; D, dorsal; V, ventral. n, number of animals analyzed.