Fig 2: Dynamics of p63 binding during development in zebrafish embryos. a Picture illustrating zebrafish developmental stages in which most experiments were performed. b Heatmaps of the 30,597 p63 binding sites (BSs) obtained from ChIPmentation in 80% of epiboly, 24 and 36 hpf stages (n = 2 biological replicates per stage). Peaks were clustered using k-means clustering, obtaining four groups: cluster a (n = 656), cluster b (n = 3,956), cluster c (n = 3,801) and cluster d (n = 22,184). Average profiles of cluster b, which was used as the group of early p63 BSs, and cluster c, as late p63 BSs, are shown on top. c Top-10 enrichment of WT expression patterns of the genes corresponding to early (top) and late (bottom) p63 BSs. The -log10 of p-value for each term is shown. d Motif enrichment analysis of the early (top) and late (bottom) p63 BSs. Five representative motifs of the top-10 were chosen. Motif logos are represented with their position in the top-10, the transcription factor (TF) name and the enrichment p-value in a color scale. e Heatmaps of the early (top) and late (bottom) p63 BSs clustered according to chromatin marks H3K4me3, H3K4me1 and H3K27ac in 80% of epiboly and 24 hpf. Peaks were clustered using k-means clustering, obtaining three sub-clusters per each group: b1 (n = 875), b2 (n = 912), b3 (n = 2,169), c1 (n = 396), c2 (n = 330) and c3 (n = 3,075). f Box plots showing the distance of the peak sub-clusters from (e) to the transcription start sites (TSS) of the nearby genes. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. Source data are provided as a Source Data file. g Motif enrichment analyses of the peak sub-clusters from (e) representing the most enriched motif in each one, their motif logos, percentage of peaks containing the motif and associated enrichment p-value. See also Supplementary Fig. 1

Fig 3: 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 4: Caspase-dependent proliferation after stem cell ablation. a Schematic of a 4-day post-fertilization (dpf) zebrafish larvae. Large region denotes area of the animal where cell death and proliferation were quantified before and after cell ablation. Small region marks the area used for fixed and live imaging. b Timeline for the addition and removal of metronidazole (MTZ). c The zc1036a GAL4 enhancer trap line drives expression of fluorescently tagged nitroreductase (NTR) in a subset of p63-positive basal stem cells (scale = 100 µm, 50 µm inset). Maximum intensity projections of confocal images for activated caspase-3 (d–f) and bromodeoxyuridine (BrdU) (g–i) at different points after inducing damage (scale = 50 µm). j Quantification of active caspase-3- and BrdU-positive cells reveals a temporal relationship for the proliferative response. Mean number of positive cells from at least n = 11 individual larvae across three independent experiments per time point are plotted. NT=No treatment. k Mean number of BrdU-positive cells in individual larvae after induced apoptosis (n = 31) and in combination with treatment of the apoptosis inhibitor, NS3694 (AI) (n = 77) or caspase-3 peptide inhibitor zDEVD-fmk (zDEVD) (n = 49). Data are from three independent experiments and error bars represent sd; ****p < 0.0001. One-way analysis of variance (ANOVA) with Dunnett’s mutiple comparisons test (j, k)

Fig 5: Zebrafish tp63-/- mutant embryos show de-regulated epidermal program. a Whole-mount embryo immunostaining of p63 in wild-type (WT), heterozygous tp63+/- and homozygous tp63-/- embryos at 32 h post fertilization (hpf) showing the absence of p63 expression in tp63-/- mutants. Left, zoomed lateral view of the tail; middle, lateral view of whole-embryo; right, zoomed lateral view of the yolk. YSL, yolk syncytial layer. b Whole-mount in situ hybridization of the tbx5a gene in WT and tp63-/- embryos at 36 hpf showing expression in eye, heart and pectoral fin bud, the latter one being reduced in tp63-/- mutant accordingly with the absence of this anatomical structure. Left, lateral view of whole embryo; middle, zoomed lateral view of the pectoral fin bud; right, dorsal view of whole embryo. For a and b, anterior is to the right. For a and b, scale bars represent 250 µm. c Differential analysis of gene expression between WT and tp63-/- at 36 hpf from RNA-seq (n = 3 biological replicates per condition). The log2 base mean WT transcript expression levels versus the log2 fold-change of expression are plotted. Transcripts showing a statistically significant differential expression (p < 0.05) are highlighted in red (upregulated) or blue (downregulated). d Box plot representing the absolute log2 fold-change of the up- (n = 973) and down-regulated (n = 1,371) transcripts from (c). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. p value according to the Wilcoxon’s rank sum test is shown. Source data are provided as a Source Data file. e Gene Ontology (GO) Biological Process term enrichment of the genes corresponding to the up- and down-regulated transcripts from (c). f Top-10 enrichment of WT expression patterns of the genes corresponding to the up- and down-regulated transcripts from (c). For e and f, the -log10 of p-value for each term is shown