A Dominant-Negative Form of p63 Is Required for Epidermal Proliferation in Zebrafish  Hyunsook Lee, David Kimelman  Developmental Cell  Volume 2, Issue 5, Pages 607-616 (May 2002) DOI: 10.1016/S1534-5807(02)00166-1

Figure 1 ΔNp63α is Highly Conserved between Zebrafish and Mouse The amino acid sequence alignment between zebrafish and mouse ΔNp63α is shown. The DNA binding domain, oligomerization domain, SAM domain, and a potential transcription inhibition domain are underlined. DBD, DNA binding domain; OD, oligomerization domain; SAM, sterile alpha motif; TID, transcription inhibition domain. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 2 ΔNp63 Expression Profile (A) RT-PCR analysis of ΔNp63 at different time points after fertilization. M is a 100 bp ladder. Numbers denote the hours after fertilization (hpf). β-actin was amplified from the same samples as a control. The ΔNp63 cDNA was used as a positive control in the ΔNp63 RT-PCR. Note that ΔNp63 expression is detected from 5 hpf soon after the start of zygotic gene expression which begins at 3 hpf. (B–F) ΔNp63 protein was detected with a mouse monoclonal antibody (Yang et al., 1998) using whole-mount embryo immunohistochemistry. (B and C) Expression at 10.5 hpf. ΔNp63 was detected in the skin, eye, and somites (arrows). (B) shows a side view; (C) shows a dorsal view. (D) Side view of a 23 hpf embryo. Note that ΔNp63 is exclusively in the nucleus of the eye and fin folds (arrows), which is not observed in the earlier stages. (E and F) 49 hpf embryos. (E) shows ΔNp63α expression in the fin folds in the posterior region. (F) shows a dorsal view of the anterior region. Note the ΔNp63 expression in the forming pectoral fins (arrows). (G) 49 hpf embryos examined by confocal microscopy in the region of the pectoral fin bud. ΔNp63 was detected with a secondary antibody coupled to Alexa Fluor 488 (green), and the embryos were counterstained with propidium iodide (red). The ΔNp63 expression is restricted to the nuclei in the outermost layers of the fin bud and the epidermis covering the body, but is not found in the underlying mesenchyme. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 3 Specific Knockdown of ΔNp63 Expression by Two Different Morpholino Oligonucleotides (A) The two morpholino oligonucleotides used in this study (MO I and MO II) are marked in the 5′ untranslated region of ΔNp63. M denotes the translational start site. (B–G) Wild-type and morphant embryos at 49 hpf. (B)–(D) show the growing fin fold in the posterior region of the embryo. Black lines mark the outer boundary of the fin fold. (E) and (G) show the dorsal view with an arrow marking the developing pectoral fin in uninjected embryo and the lack of outgrowth of the fin bud in the morpholino-injected embryos. Both MO I (C and F) and MO II (D and G) show an absence of pectoral fin and fin fold compared with uninjected (B and E) embryos. (H and I) Confocal image of embryos at 26 hpf stained with the anti-p63 antibody and detected by secondary antibody conjugated to Alexa Fluor 568. (H) shows an uninjected embryo, whereas (I) shows an embryo injected with MO I, demonstrating that the morpholino eliminates p63 expression. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 4 Disruption of Fin and Epidermis by Anti-p63 Morpholino Oligonucleotides Morpholino (MO II)-injected embryos (B, D, and F) are compared with uninjected embryos (A, C, and E) at 23 hpf (A and B) and 3 dpf (C–F). (A and B) Side view of 23 hpf embryos is shown. (C and D) Dorsal view of anterior region is shown. (E and F) Posterior region of the developing embryos at 3 dpf is shown. Outer boundary of the growing fin fold is marked with black lines. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 5 ΔNp63 Is Absolutely Required for Epidermal Proliferation (A) ΔNp63 marks the proliferating cells in the developing epidermis. Wild-type embryos at 49 hpf were labeled with BrdU (5′-bromodeoxyuridine) in vivo and processed for immunohistochemistry with an anti-BrdU antibody conjugated to FITC (Aa, green). ΔNp63 was detected by immunostaining with the anti-p63 antibody and a secondary antibody conjugated to Alexa 568 (Ab, red). (c) Shows the merged image. Yellow dots represent cells positive for both ΔNp63 and BrdU. Note that all Np63-positive cells also stain for BrdU. To produce the images shown here, 27 confocal images were taken at different levels and incorporated. (B) Loss of Proliferating Epithelial Cells in ΔNp63 Morphants. (a–d) Wild-type and MO II-injected embryos at 49 hpf were labeled with BrdU and stained with an anti-BrdU FITC-coupled antibody. Injection of the morpholino oligonucleotide MO II (b and d) eliminated the BrdU labeling seen in the skin and apical fold in uninjected embryos (a and c). The yolk in the embryos is autofluorescent. (e and f) A TUNEL assay shows that apoptosis is not increased by the morpholino injection. Green dots in the upper left of panels of (e) and (f) are TUNEL-positive cells. The staining in the lower right of the MO II-injected embryo (Bf) is autofluorescence. Both panels are views of the posterior region of the embryo. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 6 ΔNp63α Acts As a Dominant-Negative Inhibitor of p53-Responsive Transcriptional Activation (A) Transactivation by p53 is inhibited by ΔNp63α in a dose-dependent manner in cell culture. The effect of zebrafish ΔNp63α on p53 transcription activation was tested in BHK cells transfected with a p53 reporter construct (PG13-CAT). Zebrafish ΔNp63α, like a dominant-negative mutant p53 R273L (Hollstein et al., 1991; Lee et al., 1995, 1999), had no effect on p53-mediated transcriptional activation, while wild-type p53 activated the reporter activity 10-fold. p53 activity was inhibited in a dose-dependent manner by overexpression of ΔNp63α in a p53:ΔNp63α ratio of 1:1, 1:2, and 1:3. Values on the y axis are O.D. 405–490 from the CAT ELISA assay (Roche Biochemicals), representing the relative reporter activity. (B) ΔNp63α is a potent inhibitor of p53-mediated transcriptional activation in zebrafish embryos. PG13-CAT activity was measured in zebrafish embryos injected with the reporter plasmid and p53-GR or ΔNp63α-GR as shown. Endogenous reporter activity, which is likely to be due to p53 or a related family member, was inhibited by overexpression of ΔNp63α-GR. Overexpression of p53-GR increased the reporter activity, and this was markedly inhibited by coexpression of ΔNp63α-GR. Dexamethasone was added after the injection. Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)

Figure 7 Interplay between ΔNp63α and p53 Determines Epidermal Proliferation in Developing Embryos (A–C) 2-day-old embryos are shown. Overexpression of p53-GR results in a lack of fin fold outgrowth in embryos induced with dexamethasone 14 hr postfertilization (B), compared with untreated embryos injected with p53-GR (A). The defects caused by p53 overexpression were rescued by coexpression of p53-GR and zebrafish ΔNp63α-GR (C). (D–F) 3-day-old embryos are shown. (D) Overexpression of p53-GR resulted in embryos lacking their epidermis. (E and F) This defect was not seen in embryos coexpressing p53-GR and ΔNp63α-GR. Dexamethasone was added 14 hr postfertilization to the embryos shown in (B)–(F) (Bakkers et al., 2002). Developmental Cell 2002 2, 607-616DOI: (10.1016/S1534-5807(02)00166-1)