Fig 2: Tp63 positive cells accumulate at the nascent fin fold in sibling but not mutant embryos.Embryos were incubated with the anti-Tp63 antibody, and then co-stained with DAPI. (A) Embryos were collected at the indicated stages. Note the increase in Tp63-positive cells on the dorsal and ventral midline as the embryos age. (B–D) The Tp63-positive presumptive epidermis at the 18-somite stage (B) is one layer thick, but increases to multiple layers by the 24-somite stage in sibling (C) but not in yap1;wwtr1 double mutant embryos (D). Confocal images of the midline of the tailbud, with anterior to the left. The zoomed in images in panels B-D are of the dorsal side, and the arrows in the merged images point to the rare presumptive epidermis Tp63-negative cells.

Fig 3: Expansion of Tp63 expression across the layers of wound epidermis in fins treated with the BMP inhibitor during regeneration. (A and B) Immunofluorescence staining of fin sections at 4 dpa with antibodies against chondroitin sulphate (green) to visualize lepidotrichia, and Tp63 (red), a marker of the basal epithelium. DAPI (blue) demarcates nuclei. Orange dashed lines show the amputation plane, white dashed lines underly the basal wound epithelium. (C) A schematic representation of the fin outgrowth with ladder-like brown lines on each side of the wound epidermis, indicating the approximate positions along which the measurements were performed for quantification of nuclear layers. (D) Quantification of nuclei along the width of the wound epidermis as illustrated in (C). N = 3 fins (control) N = 4 (LDN193189-treated fins); three ray sections per fin; error bar SEM; n.s., not significant, *p < 0.05; unpaired two-tailed Student’s t-test.

Fig 4: A schematic summary of the phenotype caused by the inhibition of BMP signaling during outgrowth formation following fin amputation in platyfish. The schematic is based on immunofluorescence staining of fin sections at 4 dpa. Pharmacological inhibition of BMP signaling prevents regeneration of actinotrichia and lepidotrichia. Furthermore, the nuclear marker of the basal epidermal layer, Tp63, is abnormally detected throughout all layers of the wound epidermis in LDN193189-treated fins, suggesting an abnormality in the differentiation of this tissue.

Fig 5: p63 binding to epidermal enhancers precedes chromatin opening. a Alluvial plot showing the transition between 80% of epiboly and 24 hpf stages of the 22,583 peaks showing p63 binding (blue), chromatin accessibility (ATAC; red) or both (purple). Peaks showing no p63 binding in any of the two analyzed developmental stages were not considered. The transitions lead to eight groups of p63 BSs: group 1 (n = 332 peaks), group 2 (n = 145 peaks), group 3 (n = 43 peaks), group 4 (n = 1,284 peaks), group 5 (n = 3,619 peaks), group 6 (n = 1,227 peaks), group 7 (n = 896 peaks) and group 8 (n = 14,037). b Heatmaps of p63 and ATAC-seq signal in 80% of epiboly and 24 hpf stages for the groups resulting from the alluvial plot transitions in (a). Groups 2 and 3 were omitted due to their small number of peaks. c Heatmaps and average profiles of H3K4me3, H3K4me1 and H3K27ac in 80% of epiboly and 24 hpf for the group 4 of peaks from (a) (pioneered p63 BSs). d Motif enrichment analysis of the pioneered p63 BSs. The top-5 motifs are represented with their motif logos, position in the top-5, the TF name and the enrichment p-value in a color scale. e Top-10 enrichment of WT expression patterns for the genes corresponding to the pioneered p63 BSs. The -log10 of p-value for each term is shown. f Genome tracks of p63 ChIP-seq, ATAC-seq, H3K27ac, H3K4me1, and H3K4me3 in the indicated developmental stages showing signal intensities in a region of chromosome 23. Pioneered p63 BSs associated to the epidermis-expressed gene lama5 are highlighted in light orange. The Genes track represents ENSEMBL annotated genes. See also Supplementary Fig. 4