Sequential formation of epibranchial cartilages posteriorly to anteriorly. (A–I) Ventral views of dissected facial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone). (A’–I’) Cropped images of one side of the corresponding A-I. The range of SL at each stage of Eb development is marked below. (A,A’) Five pairs of Cbs are seen, with the dorsal ends of five Cbs on one side marked with dots. (B,B’) A tiny piece of Eb4 at the dorsal end of the fourth Cb is indicated by black arrowheads, with the posterior dorsal tip of the fourth Cb marked with red arrowheads. (C,C’) Black arrowheads indicate growing Eb4. AECb4 is evident at the dorsal end of the fourth Cb and marked with red arrowheads. (D,D’) The emerging Eb3 at the dorsal end of the third Cb is marked with black arrowheads, with the Eb4 and AECb4 indicated by grey and red arrowheads, respectively. (E,E’) Black arrowheads mark growing Eb3, with grey and red arrowheads indicating Eb4 and AECb4, respectively. (F,F’) Eb2 appearing at the dorsal end of the second Cb is indicated by black arrowheads, along with Ebs 3 and 4 marked with grey arrowheads. Red arrowheads indicate AECb4. (G,G’) Black arrowheads indicate growing Eb2, with grey and red arrowheads indicating Ebs 3 and 4 and AECb4, respectively. (H,H’) Eb1 at the dorsal end of the first Cb is marked with black arrowheads, with Ebs 2-4 and AECb4 indicated by grey and red arrowheads, respectively. (I,I’) Black arrowheads mark growing Eb1, with growing Ebs 2-4 and AECb4 indicated by grey and red arrowheads. (J) Timeline of the development of the epibranchial cartilages. Dpf indicates the number of days post-fertilization to reach the corresponding SL, with “n” indicating the number of fish examined at each SL. The number in the colored box is the number of fish in the same SL at the corresponding developmental stages. AECb4 formation that seems to coincide with Eb4 formation is not scored. (K,K’) Ventral views of dissected facial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 8 mm SL. Black arrowheads indicate 4 Ebs, with red and grey arrowheads indicating AECb4 and 2 Pbs, respectively. Each facial skeletal element is labeled. (K’) Cropped image of one side of (K). Anterior is to the left. Dorsal is at the top in (A’–K’). Cb, ceratobranchial cartilage; Eb, epibranchial cartilage; AECb4, accessory element of the ceratobranchial 4; Pb, pharyngobranchial cartilage; SL, standard length; A, anterior; P, posterior; D, dorsal; V, ventral.

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.

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.

Association of the dorsal end of ceratobranchial cartilage with epibranchial cartilage formation. (A–F) Ventral views of dissected facial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 8.0 mm SL. Each facial skeletal element is labeled. Anterior is to the left. (A) In wild types, black and red arrowheads, arrows, and grey arrowheads indicate five Cbs, 4 Ebs, and 2 Pbs on one side. Red arrowheads mark the dorsal ends of the fifth Cbs, which completely ossify (red), with no Alcian Blue-positive cartilage (blue) present. (B) In a pax1a mutant, a hypoplastic Cb2 that loses the dorsal region is marked with an arrowhead. A dorsal piece of the hypoplastic Cb1, with the corresponding Eb, is indicated by a black arrow. Red arrow marks a ventral piece of the hypoplastic Cb1. (B’) A high-magnification image of the left side of the pax1a mutant in (B). Dorsal is at the top. (B”) A high-magnification image of the right side of the pax1a mutant in (B), with a cropped image of the dorsal piece of hypoplastic Cb1 and the corresponding Eb1 in the lower right corner. Dorsal is at the bottom. (C) Black arrowheads, arrows, and grey arrowheads indicate four Cbs, 3 Ebs, and 2 Pbs on the left side of the compound animals transiently expressing a dominant negative form of Fgfr1 receptor (DN-Fgfr1) in the nkx2.3-positive pharyngeal endoderm. On the left side, Cb4 and the corresponding Eb4 are missing. On the right side, five Cbs and 4 Ebs form normally. (C’) A high-magnification image of the left side of the compound animal in (C). Dorsal is at the top. (D) On the right side of a fgf10 mutant, the respective ventral and dorsal regions of the fused Cbs 4 and 5 are indicated by black and red arrowheads, with an arrow marking the corresponding Eb4. On the left side, five Cbs and 4 Ebs form normally. (D’) A high-magnification image of the right side of the fgf10 mutant in (D). Dorsal is at the bottom. (E) Black and red arrows indicate the respective dorsal and ventral pieces of hypoplastic Cb3 on the right side of the compound animal bearing Tg(nkx2.3:Gal4VP16) and Tg(UAS:DN-EphB4a) transgenes. On the left side, five Cbs and 4 Ebs form normally. (E’) A high-magnification image of the right side of the compound animal in (E), with a cropped image of the dorsal piece of hypoplastic Cb3 and the corresponding Eb3 in the lower right corner. Dorsal is at the bottom. (F) On the right side of the compound animal transiently expressing DN-EphB4a in nkx2.3-positive pouch endoderm, black and red arrowheads mark the respective ventral and dorsal regions of the fused Cbs 4 and 5, with an arrow marking the corresponding Eb-like cartilage. On the left side, five Cbs and 4 Ebs form normally. (F’) A high-magnification image of the right side of the compound animal in (F), with a cropped image of the dorsal region of the fused Cbs 4 and 5 and the corresponding Eb-like cartilage in the lower right corner. Dorsal is at the bottom. Cb, ceratobranchial cartilage; Eb, epibranchial cartilage; Pb, pharyngobranchial cartilage; AECb4, accessory element of the ceratobranchial 4; Cb1v, ventral region of Cb1; Cb1d, dorsal region of Cb1; Cb3v, ventral region of Cb3; Cb3d, dorsal region of Cb3; Cb4/5, fused Cbs 4 and 5; D, dorsal; V, ventral. n, number of animals analyzed.

Requirement of Fgf3 in epibranchial cartilage development. (A–D) Fluorescence in situ hybridization of fgf3 (green) or sox9a (green) in conjunction with the GFP immunohistochemistry (red) in Tg(sox10:GFP) reporter lines. (A’–D’) Green channel only with the Cbs outlined. Scale bar: 40 μm. Anterior is to the left. Dorsal is at the top. (A,A’)fgf3 expression in 3.5 mm SL wild-type zebrafish. Arrowheads indicate fgf3 expression in the middle and dorsal regions of Cb4 and the dorsal region of Cb3. (A’) * marks fgf3 expression in the adjacent regions to the ventral area of Cb3. (B–D)sox9a expression in wild types (B) and fgf3 mutants (C,D) at 108 hpf and 3.8 mm SL. Arrowheads mark sox9a expression in the dorsal areas of Cbs 2-5 in wild types (B) and at the ends of distorted Cbs in severe (C) and intermediate (D)fgf3 mutants. In wild types, sox9a expression is not seen in Cb1 (B). In severe fgf3 mutants, Cb1 is missing (C). In intermediate fgf3 mutants, Cb1 forms, but with no sox9a expression seen (D). (E–H) Craniofacial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone). Arrows indicate Cbs, with black arrowheads marking Ebs. AECb4 is marked with red arrowheads. (E’–H’) Cropped images of the branchial basket corresponding to (E–H). (E,E’) In 24 out of 40 wild-type siblings, 4 Ebs on each side bud off at the dorsal end of the corresponding Cbs, along with AECb4 appearing on the posterior to Eb4 at 5.5 mm SL. The remaining 16 animals form 3 Ebs on each side, yet no Eb1. (F,F′) In all 6 5.5 mm SL fgf3 mutant siblings, Cb1s are relatively normal, with the other Cbs being fused or missing. Eb1 is not observed at the dorsal end of the corresponding Cb1, with no Ebs seen at the dorsal end of fused Cbs. (G,G’) In 81 out of 91 wild-type siblings treated with heat shock at 3.5 dpf for 3 days, 4 Ebs on each side form at the dorsal end of the corresponding Cbs at 9 dpf. The remaining 10 animals form 3 Ebs on each side, yet no Eb1. All 91 animals develop 5 Cbs with normal shapes. (H,H’) In 47 out of 105 Tg(hsp70I:DN-Fgfr1) animals treated with heat shock at 3.5 dpf for 3 days, no Ebs form at the dorsal end of the corresponding Cbs at 9 dpf. 44 animals form 2 Ebs (Ebs 1 and 2), without Ebs 3 and 4. The remaining 14 fish display 3 Ebs (Ebs 1-3 or Ebs 1, 3, and 4) but no Eb 2 or 4. In all 105 fish, 5 Cbs with normal shapes form. Cb, ceratobranchial cartilage; Eb, epibranchial cartilage; AECb4, accessory element of the ceratobranchial 4; D, dorsal; V, ventral. Anterior is to the left. n, number of animals analyzed.

Requirement of fgf3 in proliferating chondrocytes in the dorsal area of ceratobranchial cartilage 4 in epibranchial cartilage 4 formation. (A–F) BrdU (red) and DAPI (blue) staining relative to sox10-positive Cbs (green) in 3.8 mm SL wild-type siblings and fgf3 mutants. Cbs are outlined in (A’,D’). (A’–F’) Red and blue channels only. (B”, C”, E”, F”) Blue channel only. (A,A’) Confocal z-stack projections showing BrdU-positive dots in wild types. All 20 animals show BrdU-positive dots in GFP-positive cells of the dorsal area of Cb4 (arrowheads). (B–C”) Confocal single-section images isolated from (A) that focus on the dorsal area of Cb4. Arrowheads indicate BrdU-positive dots colocalizing with DAPI and GFP in the dorsal region of Cb4. (D,D′) Confocal z-stack projections showing BrdU-positive dots in fgf3 mutants. 12 out of 17 animals do not show BrdU-positive dots in GFP-positive cells of the dorsal areas of distorted Cbs, with 4 of them showing BrdU-positive dots in GFP-negative cells of the adjacent regions to the dorsal areas of distorted Cbs (arrowheads). The remaining 5 animals display BrdU-positive dots in GFP-positive cells of the dorsal regions of the distorted Cbs. (E–E”) A confocal single-section image isolated from (D) that focuses on the dorsal area of the distorted Cb. No BrdU-positive dots are observed in the GFP-positive dorsal regions of the distorted Cbs. (F–F”) A confocal single-section image isolated from (D) that focuses on BrdU staining in the adjacent region of the dorsal area of the distorted Cb. Yellow arrowhead indicates a BrdU-positive dot colocalizing with DAPI but not with GFP in the adjacent dorsal region of the distorted Cb. White arrowhead marks BrdU-positive staining that does not colocalize with DAPI and GFP in the adjacent dorsal region of the distorted Cb. (G) Quantification of the number of BrdU-positive dots colocalizing with DAPI and GFP in the dorsal region of Cbs analyzed in (A,D). Data is represented on a scatter plot. **** shows p < 0.0001. Cb, ceratobranchial cartilage; D, dorsal; V, ventral. Anterior is to the left. Dorsal is at the top. Scale bar: 40 μm. n, number of animals analyzed.

A model for Fgf3-dependent epibranchial formation. The expression of fgf3 appears in the dorsal and intermediate domains of monolayered ceratobranchial cartilage before epibranchial cartilage formation begins. This fgf3 expression would promote the proliferation of chondrocytes in the dorsal area of ceratobranchial cartilage to lead to stratification. Some of the chondrocytes from the stratified dorsal end would migrate to form an epibranchial bud. Cb, ceratobranchial cartilage; Eb, epibranchial cartilage.