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. 2021 Jan 27;10:e63595. doi: 10.7554/eLife.63595

Figure 4. In vivo validation of Foxc1-dependent cartilage enhancers.

(A) Genomic regions (gene loci and GRCz10 coordinates listed) for enhancer testing on the left and GFP expression driven by the indicated peaks in stable transgenic zebrafish at 6 dpf on the right. Peaks (p) tested are shown, with Foxc1-dependent regions in purple and Foxc1-independent elements in green. μATACseq reads are shown in each row, with chondrocyte and non-chondrocyte peaks from 72 hpf embryos. Confocal projections show cartilages of the first two arches in lateral view with anterior to the left. Arrows indicate enriched expression at joint regions, and arrowheads denote relative lack of expression. (B) Confocal projections show selective loss of sox10_p2:EGFP, ucmab_p1:EGFP, and epyc_p1:EGFP transgene expression in the dorsal cartilage domains (dashed outlines) of foxc1a−/−; foxc1b−/− mutants at 72 hpf. (C) Confocal projections of in situ hybridization show selective loss of sox10, lect1, col9a3, and epyc in the dorsal cartilage domain (dashed outline) of foxc1a−/−; foxc1b−/− mutants at 56 hpf. Numbers indicate proportion of embryos in which the displayed patterns were observed. bh, basihyal; DNCCs, dorsal CNCCs; jj, jaw joint; hj, hyoid joint; hm-op, hyomandibular-opercular joint; hm-otic, hyomandibular-otic junction; hm-sy, hyomandibular-symplectic junction; M-M, Meckel’s–Meckel’s joint. Scale bars = 100 μm.

Figure 4.

Figure 4—figure supplement 1. Additional in vivo validation of cartilage-enriched accessible elements.

Figure 4—figure supplement 1.

(A) Schematic of the reporter construct for enhancer testing. Tol2, transposase integration site. E1b, minimal promoter. GFP, green fluorescent protein. pA, polyadenylation sequence. CFP, cerulean fluorescent protein. (B) Snapshots of genomic regions (gene loci and GRCz10 coordinates listed) for enhancer testing. Peaks (p) tested are shown, with Foxc1-dependent regions in purple and Foxc1-independent elements in green. μATACseq reads are shown in each row for the experiments indicated, with chondrocyte and non-chondrocyte peaks from 72 hpf embryos. DNCCs, dorsal CNCCs. (C) GFP expression (yellow) driven by the indicated peaks in stable transgenic zebrafish at 6 dpf. Confocal projections of the cartilages of the first two arches are shown in lateral view for acan_p1, slc35d1a_p1 and sparc_p1 and ventral view for col9a1a_p1. For acan_p1, sox10:Dsred labels all chondrocytes for reference in magenta. Arrows indicate expression in cartilages. sparc_p1 also drives expression in ligamentocytes and osteoblasts, and slc35d1a_p1 in pharyngeal muscles. (D) Confocal sections of representative embryos injected with enhancer constructs are shown in lateral view at 6 dpf. Mosaic expression in chondrocytes (yellow, arrows) is shown, with DIC (white channel) providing embryo context. Numbers indicate proportion of embryos positive for the co-selectable lens:CFP marker (also on the injected plasmid) in which the displayed patterns were observed. Scale bar = 100 μm.
Figure 4—figure supplement 2. In vivo validation of accessible elements from Group I.

Figure 4—figure supplement 2.

(A) Genomic regions (gene loci and GRCz10 coordinates listed) for enhancer testing. Peaks (p) tested are shown in green, and μATACseq reads are shown in each row. DNCCs, dorsal CNCCs. (B) GFP expression (yellow) driven by the indicated peaks at 36 hpf and 6 dpf. For emx3_p1 and prrx1a_p1, confocal projections of whole arch CNCCs of stable transgenic zebrafish are shown in lateral view at 36 hpf. For satb2-p1, confocal section of representative injected embryo is shown in lateral view at 6 dpf, with DIC providing embryo context in white. Numbers indicate proportion of embryos positive for the co-selectable lens:CFP marker (also on the injected plasmid) in which the displayed patterns were observed. Scale bar = 100 μm.