Fig. 4

Fig. 4 Generation and validation of a scxa:creERT2 transgenic line.

a HCR double in situ hybridization of scxa and cre at 4 days post-fertilization (dpf) shows overlap in expression in craniofacial tendons and ligaments. l, lateral ligament; sh, sternohyoidus tendon; hh, hyohyal tendon. Scale bar, 50 µm. b Schematic of 4OH-T labeling experiment to test tendon labeling in scxa:creERT2; ubi:zebrabow larvae. c, d Representative confocal images from the 4OH-T labeling validation experiment in (b). The labeled SH tendon is shown in c and labeled myoseptal cells in the trunk region are shown in d at 96 h post-fertilization (hpf). Scale bar, 50 µm. e Representative 2-photon image of 4OH-T labeling in the 3-month-old adult MST (outlined in white dotted line). Asterisks denote autofluorescent pigment in the skin. Scale bar, 100 µm. f Confocal image of anti-GFP immunofluorescence to detect CFP+ and/or YFP+ scxa-lineage cells (green) combined with RNAscope in situ hybridization of scxa (magenta). Scale bar, 100 µm. g, h Higher magnification (40x) confocal image of the tendon-bone attachment region of the MST from panel f. scxa-lineage cells overlayed with scxa signal are shown in g, while scxa-lineage cells are shown in h. Scale bar, 50 µm. i, j Higher magnification (40x) confocal image of the midbody region of the MST from panel (f). scxa-lineage cells overlayed with scxa signal are shown in (i), while scxa-lineage cells are shown in (j). Scale bar, 50 µm. k Quantification of the efficiency of scxa:creERT2 labeling in 3 independent adult zebrafish (measuring the percentage of CFP+ and/or YFP+ cells (in green) out of total scxa-expressing cells (in magenta) as detected by RNAscope in situ hybridization). Means of quantified sections from each fish are listed above and error bars in the bar graph denote the standard deviation.