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.
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