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Fig. 2 A deeply conserved Pou3f3 enhancer differentially regulates arch expression in bony and cartilaginous fish. (A) A conserved regulatory element downstream of Pou3f3 genes. ATAC-seq data from zebrafish fluorescence-activated cell sorting-purified fli1a:EGFP; sox10:DsRed double-positive arch neural crest cells (NCCs) and double-negative non-arch cells (Top), human in vitro-derived neural crest-like cells (data from ref. 15) (Middle), and mouse hyoid arch cells versus early neural crest progenitors (data from ref. 14) (Bottom). Data were visualized with the UCSC Genome Browser, and analyzed peaks are indicated. (B) mVista alignment (60, 61) of conserved 1B sequences relative to Hs1B. (C) Structure of reporter construct. (D) Compared with a pou3f3b knock-in line (Left), Dr1 and 1B reporter lines show primarily arch-restricted GFP expression at 36 hpf (lateral perspective; arches are numbered), with an additional domain of expression in the anterior head (arrowheads) as indicated. (E and E′) At 6 to 7 dpf (ventral perspective), Dr1 and bony vertebrate 1B sequences drive robust expression in the hyoid opercular mesenchyme (Op), with nominal expression in the more posterior arches, while cartilaginous fish 1B reporters induce expression evenly across the hyoid and posterior gill-bearing arches. Expression expands into the ventral operculum in the human and gar reporters (dotted line). Images in E′ are cropped subsets highlighting the posterior arches. sox10:DsRed labels neural crest-derived and otic cells at 36 hpf and chondrocytes at 7 dpf. enh., enhancer; kb, kilobase pairs; min. prom., minimal promoter; mx, maxillary expression; prom., promoter. (Scale bars, 50 µm.)