Files in this Data Supplement:
Fig. S1. Transplantation protocol.
Fig. S2. Early pouching appears normal in fras1 mutants. (A) Confocal section of WT pharyngeal arches, with pre-cartilage cells labeled using sox9a:EGFP and epithelial nuclei labeled using anti-P63; shown anterior to left, dorsal up. Yellow lines indicate section levels for B-E. (B-E) Four confocal transverse sections through arches one and two, reconstructed from a confocal stack of the embryo imaged for A, shown lateral to the left, dorsal up. (B′-E′) Epithelial morphology. At 36 hpf a tube of endoderm lies medial to the length of arches; early-p1 separates arch one and two mesenchyme dorsally, and posterior pouches fully separate posterior arch mesenchyme. Ec, surface ectoderm; Md, medial endoderm; p2, pouch 2; p3, pouch 3; p4, pouch 4; St, stomadeum. Scale bar: 100 µm.
Fig. S3. Overall cartilage growth, as assayed by hyomandibular length, does not differ between WT and fras1 mutants. Hyomandibular (Hm) length in WT and fras1 mutants, measured from the same fish graphed in Fig. 4. Although fras1 mutant hyomandibular length is slightly lower than WT hyomandibular length at some time points, this difference is not significant at any time point, nor collectively for the whole dataset. Measurements of Hm cartilage, and Hm cartilage precursors, were made using sox9a:EGFP. Length was measured across the hyomandibular plate, from the posterior edge of the Sy to the dorsal edge of the hyomandibular.
Fig. S4. Symplectic length per side and measurement errors. Average symplectic length measurements do not differ significantly between left and right sides of embryos. Measurement error also does not show side bias and is low compared with symplectic length. Data are from Sy length measurements presented in Fig. 7.
Movie 1. Time-lapse imaging of WT skeletal development, highlighting the initiation of symplectic extension and its subsequent elongation. Images of sox9a:EGFP-expressing fish, taken every 30 minutes from 34.5 to 79 hpf. Images are rendered projections of confocal stacks. Anterior to left, dorsal up. Scale bar: 100 µm.
Movie 2. Time-lapse imaging shows concurrent late-p1 and symplectic morphogenesis. her5:GFP expression reveals late-p1 morphogenesis, and sox10:mRFP expression shows skeletal morphogenesis, including symplectic extension and interhyal formation. Images taken every 30 minutes from 51 to 85 hpf. (A) Confocal transverse section showing lateral movement of late-p1 between the symplectic and ceratohyal cartilages. Lateral to left, dorsal up. During WT development, arches move laterally, so the plane of the symplectic has been adjusted to a fixed position along the mediolateral axis in this movie. Overall lateral movement of arches indicated by a white bar that shifts to the right because symplectic location is held constant. White arrowhead indicates movement of late-p1 relative to symplectic location. Transverse section plane is tracked manually within Volocity software. (B) Confocal sagittal section showing symplectic extension (yellow line), oriented anterior to left, dorsal up. Concurrent with symplectic extension, the interhyal cartilage also forms (yellow arrow). (C) Because symplectic extends throughout the movie, the section taken for A moves through time, as indicated by the blue line in C. Scale bar: 100 µm.
Movie 3. Time-lapse imaging of first arch skeletal development in WT and fras1te262 mutant. Confocal sections of sox9a:EGFP-expressing fish, shown anterior to left, dorsal up. Images were obtained every 15 minutes from 48 to 76 hpf. (A) During WT development, Meckel’s and palatoquadrate cartilages separate, and a cleft forms in between them (red). (B) This cleft fails to form in fras1te262 mutants, and Meckel’s remains continuous with palatoquadrate.
Movie 4. Time-lapse imaging of second arch skeletal development in WT and fras1te262 mutant, highlighting an unusually early forming fusion of symplectic to ceratohyal cartilages. Projections of confocal stacks of sox9a:EGFP-expressing fish. z-stacks were obtained every 15 minutes from 48 to 78 hpf. (A) WT and (B) fras1te262 mutants were raised and imaged concurrently in the same dish. In fras1te262, there is inappropriate movement (yellow arrow) of symplectic cartilage cells towards ceratohyal cartilage cells, eventually resulting in fusion (red arrow) between the two cartilages. Scale bar: 100 µm.
