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.
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