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<!DOCTYPE html> <html lang="en" class="grade-c"> <head> <title>Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice | Cell Death & Differentiation</title> <link rel="alternate" type="application/rss+xml" href="https://www.nature.com/cdd.rss"/> <link rel="preconnect" href="https://cmp.nature.com" crossorigin> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="applicable-device" content="pc,mobile"> <meta name="viewport" content="width=device-width,initial-scale=1.0,maximum-scale=5,user-scalable=yes"> <meta name="360-site-verification" content="5a2dc4ab3fcb9b0393241ffbbb490480" /> <script data-test="dataLayer"> window.dataLayer = [{"content":{"category":{"contentType":"original article","legacy":{"webtrendsPrimaryArticleType":"research","webtrendsSubjectTerms":null,"webtrendsContentCategory":null,"webtrendsContentCollection":null,"webtrendsContentGroup":"Cell Death & 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The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63α and/or ΔNp63α under the K5 promoter into p63−/− mice by in vivo genetic complementation. Whereas p63−/− and p63−/−;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63−/−;ΔN mice showed significant epidermal basal layer formation. Double TAp63α/ΔNp63α complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, ΔNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layer's genes (Ets-1, K1, transglutaminases, involucrin). 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xlink:href="#icon-download"/></svg> </a> </div> </div> </div> <div class="c-article-header"> <header> <ul class="c-article-identifiers" data-test="article-identifier"> <li class="c-article-identifiers__item" data-test="article-category">Original Article</li> <li class="c-article-identifiers__item">Published: <time datetime="2006-04-07">07 April 2006</time></li> </ul> <h1 class="c-article-title" data-test="article-title" data-article-title="">Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice</h1> <ul class="c-article-author-list c-article-author-list--short" data-test="authors-list" data-component-authors-activator="authors-list"><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-E-Candi-Aff1" data-author-popup="auth-E-Candi-Aff1" data-author-search="Candi, E">E Candi</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-A-Rufini-Aff1" data-author-popup="auth-A-Rufini-Aff1" data-author-search="Rufini, A">A Rufini</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-A-Terrinoni-Aff1" data-author-popup="auth-A-Terrinoni-Aff1" data-author-search="Terrinoni, A">A Terrinoni</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-D-Dinsdale-Aff2" data-author-popup="auth-D-Dinsdale-Aff2" data-author-search="Dinsdale, D">D Dinsdale</a><sup 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data-author-popup="auth-V-De_Laurenzi-Aff2" data-author-search="De Laurenzi, V">V De Laurenzi</a><sup class="u-js-hide"><a href="#Aff2">2</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-L_G-Spagnoli-Aff1" data-author-popup="auth-L_G-Spagnoli-Aff1" data-author-search="Spagnoli, L G">L G Spagnoli</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-M_V-Catani-Aff1" data-author-popup="auth-M_V-Catani-Aff1" data-author-search="Catani, M V">M V Catani</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-S-Ramadan-Aff1" data-author-popup="auth-S-Ramadan-Aff1" data-author-search="Ramadan, S">S Ramadan</a><sup class="u-js-hide"><a href="#Aff1">1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-R_A-Knight-Aff2" data-author-popup="auth-R_A-Knight-Aff2" data-author-search="Knight, R A">R A Knight</a><sup class="u-js-hide"><a href="#Aff2">2</a></sup> & </li><li class="c-article-author-list__show-more" aria-label="Show all 12 authors for this article" title="Show all 12 authors for this article">…</li><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-G-Melino-Aff1-Aff2" data-author-popup="auth-G-Melino-Aff1-Aff2" data-author-search="Melino, G" data-corresp-id="c1">G Melino<svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-mail-medium"></use></svg></a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a></sup> </li></ul><button aria-expanded="false" class="c-article-author-list__button"><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-down-medium"></use></svg><span>Show authors</span></button> <p class="c-article-info-details" data-container-section="info"> <a data-test="journal-link" href="/cdd" data-track="click" data-track-action="journal homepage" data-track-category="article body" data-track-label="link"><i data-test="journal-title">Cell Death & Differentiation</i></a> <b data-test="journal-volume"><span class="u-visually-hidden">volume</span> 13</b>, <span class="u-visually-hidden">pages </span>1037–1047 (<span data-test="article-publication-year">2006</span>)<a href="#citeas" class="c-article-info-details__cite-as u-hide-print" data-track="click" data-track-action="cite this article" data-track-label="link">Cite this article</a> </p> <div class="c-article-metrics-bar__wrapper u-clear-both"> <ul class="c-article-metrics-bar u-list-reset"> <li class=" c-article-metrics-bar__item" data-test="access-count"> <p class="c-article-metrics-bar__count">5994 <span class="c-article-metrics-bar__label">Accesses</span></p> </li> <li class="c-article-metrics-bar__item" data-test="altmetric-score"> <p class="c-article-metrics-bar__count">1 <span class="c-article-metrics-bar__label">Altmetric</span></p> </li> <li class="c-article-metrics-bar__item"> <p class="c-article-metrics-bar__details"><a href="/articles/4401926/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics <span class="u-visually-hidden">details</span></a></p> </li> </ul> </div> </header> </div> <div class="c-article-body"> <section aria-labelledby="Abs1" data-title="Abstract" lang="en"><div class="c-article-section" id="Abs1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Abs1">Abstract</h2><div class="c-article-section__content" id="Abs1-content"><p>Epidermal development requires the transcription factor p63, as p63−/− mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63<i>α</i> and/or ΔNp63<i>α</i> under the K5 promoter into p63−/− mice by <i>in vivo</i> genetic complementation. Whereas p63−/− and p63−/−;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63−/−;ΔN mice showed significant epidermal basal layer formation. Double TAp63<i>α</i>/ΔNp63<i>α</i> complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, ΔNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layer's genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.</p></div></div></section> <section aria-labelledby="inline-recommendations" data-title="Inline Recommendations" class="c-article-recommendations" data-track-component="inline-recommendations"> <h3 class="c-article-recommendations-title" id="inline-recommendations">Similar content being viewed by others</h3> <div class="c-article-recommendations-list"> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41467-020-16475-3/MediaObjects/41467_2020_16475_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41467-020-16475-3?fromPaywallRec=false" data-track="select_recommendations_1" data-track-context="inline recommendations" data-track-action="click recommendations inline - 1" data-track-label="10.1038/s41467-020-16475-3">Chronic expression of p16<sup>INK4a</sup> in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__access-type">Open access</span> <span class="c-article-meta-recommendations__date">01 June 2020</span> </div> </div> </article> </div> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41389-020-00261-3/MediaObjects/41389_2020_261_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41389-020-00261-3?fromPaywallRec=false" data-track="select_recommendations_2" data-track-context="inline recommendations" data-track-action="click recommendations inline - 2" data-track-label="10.1038/s41389-020-00261-3">Altering MYC phosphorylation in the epidermis increases the stem cell population and contributes to the development, progression, and metastasis of squamous cell carcinoma </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__access-type">Open access</span> <span class="c-article-meta-recommendations__date">07 September 2020</span> </div> </div> </article> </div> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41420-025-02326-x/MediaObjects/41420_2025_2326_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 class="c-article-recommendations-card__heading" itemprop="name headline"> <a class="c-article-recommendations-card__link" itemprop="url" href="https://www.nature.com/articles/s41420-025-02326-x?fromPaywallRec=false" data-track="select_recommendations_3" data-track-context="inline recommendations" data-track-action="click recommendations inline - 3" data-track-label="10.1038/s41420-025-02326-x">TAp63γ is the primary isoform of TP63 for tumor suppression but not development </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__access-type">Open access</span> <span class="c-article-meta-recommendations__date">06 February 2025</span> </div> </div> </article> </div> </div> </section> <script> window.dataLayer = window.dataLayer || []; window.dataLayer.push({ recommendations: { recommender: 'semantic', model: 'specter', policy_id: 'NA', timestamp: 1739907943, embedded_user: 'null' } }); </script> <div class="main-content"> <section data-title="Main"><div class="c-article-section" id="Sec1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec1">Main</h2><div class="c-article-section__content" id="Sec1-content"><p>The skin consists of two compartments, the dermis and the epidermis. The latter is a multilayered, stratified epithelium continuously regenerated by terminally differentiating keratinocytes, a process called cornification<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Candi E, Schmidt R and Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat. Rev. Mol. Cell Biol. 6: 328–340." href="/articles/4401926#ref-CR1" id="ref-link-section-d42514748e603">1</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 2" title="Fuchs E and Watt FM (2003) Cell differentiation. Focus on epithelia. Curr. Opin. Cell Biol. 15: 738–739." href="/articles/4401926#ref-CR2" id="ref-link-section-d42514748e606">2</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 3" title="Owens DM and Watt FM (2003) Contribution of stem cells and differentiated cells to epidermal tumours. Nat. Rev. Cancer 3: 444–451." href="/articles/4401926#ref-CR3" id="ref-link-section-d42514748e609">3</a></sup> (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1a and b</a>). Recent evidence demonstrates a major role for p63,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 4" title="Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D and McKeon F (1998) p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2: 305–316." href="/articles/4401926#ref-CR4" id="ref-link-section-d42514748e616">4</a></sup> a member of the p53 family,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 5" title="Melino G, De Laurenzi V and Vousden KH (2002) p73: friend or foe in tumorigenesis. Nat. Rev. Cancer 2: 605–615." href="/articles/4401926#ref-CR5" id="ref-link-section-d42514748e620">5</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Melino G, Lu X, Gasco M, Crook T and Knight RA (2003) Functional regulation of p73 and p63: development and cancer. Trends Biochem. Sci. 28: 663–670." href="/articles/4401926#ref-CR6" id="ref-link-section-d42514748e623">6</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 7" title="Yang A, Kaghad M, Caput D and McKeon F (2002) On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 18: 90–95." href="/articles/4401926#ref-CR7" id="ref-link-section-d42514748e626">7</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 8" title="Yang A and McKeon F (2000) P63 and P73: P53 mimics, menaces and more. Nat. Rev. Mol. Cell Biol. 1: 199–207." href="/articles/4401926#ref-CR8" id="ref-link-section-d42514748e629">8</a></sup> in this process as mutations in the TP63 gene cause limb and skin defects in humans,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 9" title="Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, Smits AP, Newbury-Ecob R, Hennekam RC, Van Buggenhout G, van Haeringen A, Woods CG, van Essen AJ, de Waal R, Vriend G, Haber DA, Yang A, McKeon F, Brunner HG and van Bokhoven H (1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99: 143–153." href="/articles/4401926#ref-CR9" id="ref-link-section-d42514748e633">9</a></sup> and p63−/− mice have no epidermis, no limbs and die at birth owing to dehydration.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR and Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713." href="/articles/4401926#ref-CR10" id="ref-link-section-d42514748e638">10</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e641">11</a></sup></p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-1" data-title="Figure 1"><figure><figcaption><b id="Fig1" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 1</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/1" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig1_HTML.jpg?as=webp"><img aria-describedby="Fig1" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig1_HTML.jpg" alt="figure 1" loading="lazy" width="685" height="377"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-1-desc"><p>Expression of TAp63<i>α</i> or ΔNp63<i>α</i> under the control of the K5 promoter in transgenic mice. We obtained two different mouse lines for each isoform, all of which showed similar levels of expression of TAp63<i>α</i> or ΔNp63<i>α</i> proteins in the basal layer. (<b>a</b>) ΔNp63 and TAp63 are both expressed in the basal layer, with ΔNp63 being predominant. (<b>b</b>) The role of the p63 protein is still controversial,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 23" title="McKeon F (2004) p63 and the epithelial stem cell: more than status quo? Genes Dev. 18: 465–469." href="/articles/4401926#ref-CR23" id="ref-link-section-d42514748e673">23</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 24" title="Nicotera P and Melino G (2005) Neurodevelopment on route p63. Neuron 48: 707–709." href="/articles/4401926#ref-CR24" id="ref-link-section-d42514748e676">24</a></sup> regulating either the stem cells/transient amplifying (TA) cells<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e680">11</a></sup> (1) or their differentiation<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR and Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713." href="/articles/4401926#ref-CR10" id="ref-link-section-d42514748e684">10</a></sup> (2) or cell death<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 28" title="Jacobs WB, Govoni G, Ho D, Atwal JK, Barnabe-Heider F, Keyes WM, Mills AA, Miller FD and Kaplan DR (2005) P63 is an essential proapoptotic protein during neural development. Neuron 48: 743–756." href="/articles/4401926#ref-CR28" id="ref-link-section-d42514748e688">28</a></sup> (3). The data herein reported are compatible with the first hypothesis, with distinct roles for ΔNp63 and TAp63. (<b>c</b>) The 5.2 kb K5 constructs used to express TAp63<i>α</i> or ΔNp63<i>α</i> in basal keratinocytes. Mouse cDNAs are fused in-frame at the N-terminal end with an HA epitope. The distances in kb are indicated in the figure. (<b>d</b>) Expression of the transgene in cultured primary keratinocytes. Western blots for p63 (left, showing endogenous and transgenes) or HA tag (right, showing only transgenes). TA and ΔN indicate the protein expression in representative transgenic mice (ΔNp63<i>α</i> or TAp63<i>α</i>). The two lanes on the right show a marker control for both TAp63<i>α</i> and ΔNp63<i>α</i> proteins. (<b>e</b>) Immunofluorescence for the transgene (stained using an antibody against the HA tag) shows a nuclear localisation for both the TAp63<i>α</i> and ΔNp63<i>α</i> proteins in primary keratinocytes cultured from the transgenic mice. Bar=15 <i>μ</i>m. (<b>f</b>) Immunohistochemistry of p63 in epidermis, using anti-HA antibody for transgenic mice and anti-p63 (Ab4 clone) for wt mice, showing overexpression of the transgene (brown colour) in the basal layer of the epidermis only. Bar=120 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/1" data-track-dest="link:Figure1 Full size image" aria-label="Full size image figure 1" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>The expression of p63 proteins originates from two promoters, giving rise to TAp63 and ΔNp63 isoforms. In addition, both isoforms undergo alternative splicing at the C-terminus producing three different TAp63 and ΔNp63 isoforms (<i>α</i>, <i>β</i> and <i>γ</i>). Although there is solid evidence that p63 is involved in epithelial development,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR and Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713." href="/articles/4401926#ref-CR10" id="ref-link-section-d42514748e757">10</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e760">11</a></sup> for example, via regulation of PML,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e764">11</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 12" title="Bernassola F, Oberst A, Melino G and Pandolfi PP (2005) The promyelocytic leukaemia protein tumour suppressor functions as a transcriptional regulator of p63. Oncogene 24: 6982–6986." href="/articles/4401926#ref-CR12" id="ref-link-section-d42514748e767">12</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Keyes WM, Wu Y, Vogel H, Guo X, Lowe SW and Mills AA (2005) p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev. 19: 1986–1999." href="/articles/4401926#ref-CR13" id="ref-link-section-d42514748e770">13</a></sup> and in aging,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 13" title="Keyes WM, Wu Y, Vogel H, Guo X, Lowe SW and Mills AA (2005) p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev. 19: 1986–1999." href="/articles/4401926#ref-CR13" id="ref-link-section-d42514748e775">13</a></sup> the relative contribution of the different N-terminal isoforms to epidermal formation has not been established. To address this issue, we have, therefore, generated transgenic mice expressing either TAp63<i>α</i> or ΔNp63<i>α</i> under the control of the keratin (K) 5 promoter. The K5 promoter specifically drives the expression of the gene in the basal layer, the proliferative compartment of the epithelium. TAp63<i>α</i> and ΔNp63<i>α</i> transgenic mice were used to generate isoform-specific complemented mice in the knockout background. The data presented are consistent with a role for ΔNp63<i>α</i> in controlling the expansion of epithelial cells from progenitor precursors in epidermal epithelia, to allow TAp63<i>α</i>, acting synergistically and/or subsequently to ΔNp63<i>α</i>, to control epithelial differentiation.</p></div></div></section><section data-title="Results"><div class="c-article-section" id="Sec2-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec2">Results</h2><div class="c-article-section__content" id="Sec2-content"><h3 class="c-article__sub-heading" id="Sec3">Transgenic complemented mice</h3><p>To elucidate the individual role of the TAp63<i>α</i> and ΔNp63<i>α</i> isoforms in the development of the epidermis, we generated transgenic mice expressing either isoform under the control of the K5 promoter and then crossed these mice into a p63−/− background (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1c–f</a>). Founder transgenics (four TAp63<i>α</i> and five ΔNp63<i>α</i>) were generated by microinjecting the purified transgene into the pronuclei of zygotes and then implanting the zygotes into pseudo-pregnant female mice. Lines were established and maintained by backcrossing the founders with C57/B6 mice. All founders were fertile and produced transgenic offspring at the expected Mendelian frequencies without any obvious abnormalities. We obtained two different mouse lines for each isoform, all of which showed similar levels of expression of TAp63<i>α</i> or ΔNp63<i>α</i> proteins in the basal layer. The density of the hair follicles, and outer appearance of the skin and fur were normal. Efficient expression of the transgenes was evident both by immunostaining on skin biopsies and by Western analysis on cultured primary keratinocytes. Expression was restricted to the epidermal basal layer and in tissues where K5 is normally expressed (thymus, eye, lung), but not in other tissues including bone, muscle, liver (not shown). <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1d</a> shows the immunoblot staining with antibodies specific for p63 (left panel) and for the haemagglutin-antigen (HA) tag (right panel). The transgene was overexpressed as a nuclear protein, as shown in <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1e</a>, in the basal layer of the epidermis, <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1f</a>, consistent with the known specificity of the K5 promoter. To obtain double mutant mice, the transgenic mice overexpressing TAp63<i>α</i>/ΔNp63<i>α</i> were backcrossed with p63+/− mice.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e851">11</a></sup></p><p>The p63−/−;TA (mice knockout for p63, re-expressing TAp63<i>α</i>) and p63−/−;ΔN (mice knockout for p63, re-expressing ΔNp63<i>α</i>) transgenic mice died at birth, like the p63−/− mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig2">Figure 2</a>). TAp63<i>α</i> complemented mice, like p63−/− animals, had only very limited areas of epithelialisation with abnormal visibility of the vasculature through the skin (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig2">Figure 2d</a>) due to the absence of the epidermis. In contrast, the p63−/−;ΔN transgenic mice showed greater, although still limited, macroscopical formation of the epidermis (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig2">Figure 2c</a>). In agreement with this observation, the ΔNp63<i>α</i> complemented animals expressed greater amounts of the characteristic basal layer proteins, K5 and K14, than either the p63−/− or the p63−/−;TA mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig3">Figures 3a, b</a> and <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig4">4a</a>). No significant increase in the expression of upper layer markers (K1, loricrin) was detected in p63−/−;TA and p63−/−;ΔN transgenic mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig3">Figure 3c, d</a> and <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig4">4b, c</a>).</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-2" data-title="Figure 2"><figure><figcaption><b id="Fig2" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 2</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/2" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig2_HTML.jpg?as=webp"><img aria-describedby="Fig2" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig2_HTML.jpg" alt="figure 2" loading="lazy" width="627" height="456"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-2-desc"><p>Reintroduction of TAp63<i>α</i> or ΔNp63<i>α</i> in p63−/− mice by genetic complementation. Morphology (<b>a</b>–<b>d</b>) and haematoxylin and eosin (H&E) skin biopsy staining (<b>e</b>–<b>l</b>) of newborn mice. Bar=250 <i>μ</i>m (<b>e</b>, <b>g</b>, <b>i</b>, <b>k</b>) or 50 <i>μ</i>m (<b>f</b>, <b>h</b>, <b>j</b>, <b>l</b>). (<b>a</b>, <b>e</b>, <b>f</b>) wt newborn mice. (<b>b</b>, <b>g</b>, <b>h</b>) p63−/− knockout mice. At a gross morphological level, there is no epidermis, allowing the clear visibility of the dermis. Limbs and skin annexes are also absent. Histologically, only few skin patches are present. (<b>c</b>, <b>i</b>, <b>j</b>) p63−/− transgenic mice with reintroduction of ΔNp63<i>α</i>. There is some reformation of the epidermis, as shown by a detachable upper layer. (<b>d</b>, <b>k</b>, <b>l</b>) p63−/− transgenic mice with reintroduction of TAp63<i>α</i>. There is no epidermis at a gross morphology level; histologically, the skin is similar to the p63 null mice</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/2" data-track-dest="link:Figure2 Full size image" aria-label="Full size image figure 2" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-3" data-title="Figure 3"><figure><figcaption><b id="Fig3" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 3</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/3" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig3_HTML.jpg?as=webp"><img aria-describedby="Fig3" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig3_HTML.jpg" alt="figure 3" loading="lazy" width="685" height="361"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-3-desc"><p>Expression of epidermal differentiation proteins by confocal immunostaining in p63−/− mice before and after reintroduction of TAp63<i>α</i> or ΔNp63<i>α</i>. Colour code and markers of the basal (K5, K14) layers are indicated. Bar=50 <i>μ</i>m. (<b>a</b>) Staining for K14, p63 and Dapi. K14 was visible in very rare patches of the epidermis in the p63−/− mice. The reintroduction of ΔNp63<i>α</i> into the p63−/− background allows the reconstruction of the basal layer in several skin areas, with expression of K14. White stars indicate autofluorescent blood cells, also visible in unstained slides. (<b>b</b>) Staining for K5, p63 and Dapi. Like K14, K5 is also re-expressed in p63−/−;ΔN transgenic mice. K5 (like K14) was already visible in very few sections of the p63−/− mice, as shown. White stars indicate autofluorescent blood cells, also visible in unstained slides. Skin biopsies were taken from newborn mice. (<b>c</b>, <b>d</b>) Colour code and markers of the upper (K1, loricrin) layers are indicated. (<b>c</b>) Staining for K1, p63 and Dapi. K1 is detected in very rare areas of p63−/−, p63−/−;ΔN, p63−/−;TA complemented mice. (<b>d</b>) Staining for loricrin, p63 and Dapi. Loricrin is detected in very rare areas of upper epidermal layers in p63−/−, p63−/−;ΔN, p63−/−;TA complemented mice. White stars indicate autofluorescence of blood cells. All bars=50 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/3" data-track-dest="link:Figure3 Full size image" aria-label="Full size image figure 3" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-4" data-title="Figure 4"><figure><figcaption><b id="Fig4" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 4</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/4" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig4_HTML.jpg?as=webp"><img aria-describedby="Fig4" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig4_HTML.jpg" alt="figure 4" loading="lazy" width="316" height="587"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-4-desc"><p>Expression of epidermal differentiation proteins by Western blot in p63−/− mice before and after reintroduction of TAp63<i>α</i> and/or ΔNp63<i>α</i>. (<b>a</b>–<b>d</b>) Expression of cornification proteins in p63−/− mice after the reintroduction of TAp63<i>α</i> and/or ΔNp63<i>α</i>. E19.5 embryos were incubated for 24 h in buffer (1% SDS, 20 mM DTT) to solubilise the outermost epidermal layers; proteins were quantified, electrophoresed and blotted. Lanes: 1, p63−/−;ΔN;TA ; 2, p63−/−;TA ; 3, p63−/−;ΔN ; 4, p63−/− ; 5, wt. (<b>a</b>) K14 expression. A limited, but definite, K14 expression was already detectable in p63−/− mice. The reintroduction of the ΔNp63<i>α</i> protein (but not TAp63<i>α</i>) was sufficient to significantly increase K14 protein expression. (<b>b</b>) Western blot for filaggrin, already expressed at a low level in p63−/− mice, indicating the ability of at least some keratinocytes to differentiate and express markers of the upper layers. As for K14 (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig3">Figure 3a</a>), the reintroduction of the ΔNp63<i>α</i> protein (but not TAp63<i>α</i>) was sufficient to significantly increase the expression of filaggrin. (<b>c</b>) Western blot for loricrin. Results were similar to filaggrin. Proteins from wt mice showed either monomer, owing to the solubilisation of loricrin from the L-granules, or heavily crosslinked proteins (indicated by L-arrow, low molecular weight polymers; and H-arrow, high molecular weight polymers). All transgenic mice showed mainly intermediate oligomers and no monomer (formation of L-granules is abnormal in these mice). Again, p63−/− mice (and even more mice with reintroduction of the ΔNp63<i>α</i> protein, L-arrow) show the expression of loricrin, with a significant degree of crosslinked polymers, suggesting that at least some TG have been expressed and activated. (<b>d</b>) Loading control, actin</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/4" data-track-dest="link:Figure4 Full size image" aria-label="Full size image figure 4" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>At the ultrastructural level, unlike wild-type (wt) mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig5">Figure 5a</a>), these three groups (p63−/−, p63−/−;TA, p63−/−;ΔN) were all devoid of fully keratinised squamous corneocytes, intercellular lipid or corneodesmosomes, and there were no recognisable filaggrin granules or keratin fibrils (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig5">Figure 5b–d</a>). No traces of hemidesmosomes/basement membrane were found in any of these animals. The surface layers of KO mice were dominated by fibroblast-like cells interspersed by irregular cell profiles that contained a few randomly arranged keratin filaments. These cells were not restricted to the outer surface but showed localised cornification of their cell envelopes and many contained some enlarged, osmiophilic granules (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig5">Figure 5b</a>). The contents of these granules were extracted, from resin sections, and were identified as loricrin granules (not shown). TAp63<i>α</i> complemented mice had only very limited areas of epithelialisation and the resulting cell profiles usually contained enlarged loricrin granules together with accumulations of a moderately electron-dense material, presumably filaggrin (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig5">Figure 5c</a>). In contrast, the epidermis of the p63−/−;ΔN mice contained many cells with traces of randomly dispersed keratin filaments and signs of nuclear disintegration or cornification of the cell envelope, but these cells were often overlaid by non-cornified cells (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig5">Figure 5d</a>). Many of the cornified cells contained accumulations of the moderately electron-dense material, seen in TAp63<i>α</i> complemented mice. These data indicate that the effect of TAp63<i>α</i> in regenerating the epithelium, if reintroduced through the K5 promoter, is very limited, and that selective reintroduction of ΔNp63<i>α</i> can only partially restore the p63 null phenotype. We therefore formulated the hypothesis that a synergistic action of TAp63<i>α</i> and ΔNp63<i>α</i> is necessary for the formation of the epidermis.</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-5" data-title="Figure 5"><figure><figcaption><b id="Fig5" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 5</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/5" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig5_HTML.jpg?as=webp"><img aria-describedby="Fig5" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig5_HTML.jpg" alt="figure 5" loading="lazy" width="413" height="452"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-5-desc"><p>Ultrastructure of epithelial patches following reintroduction of TAp63<i>α</i> or ΔNp63<i>α</i> in p63−/− mice by genetic complementation showing filaggrin granules (white arrows) and loricrin granules (black arrows). (<b>a</b>) wt mouse; cornified squamous cells (top left) overlie the granular layer where numerous filaggrin granules are associated with keratin fibrils. (<b>b</b>) p63−/− knockout mouse; fibroblast-like cells overlie a cell with localised cornification of the cell envelope and enlarged loricrin granules. (<b>c</b>) p63−/− transgenic mouse with reintroduction of TAp63<i>α</i>; irregular cornified cell envelops contain enlarged loricrin granules and accumulations of a moderately electron-dense material (white arrowheads). (<b>d</b>) p63−/− transgenic mouse with reintroduction of ΔNp63<i>α</i>; flattened cells predominate but cornified cells, with accumulations of a moderately electron-dense material (white arrowheads), are distributed throughout the superficial layers. All bars=1 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/5" data-track-dest="link:Figure5 Full size image" aria-label="Full size image figure 5" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><h3 class="c-article__sub-heading" id="Sec4">Double-complemented mice</h3><p>Accordingly, we reintroduced both TAp63<i>α</i> and ΔNp63<i>α</i> into the p63−/− background (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig6">Figure 6</a>). The mice died within 12–18 h of birth and the gross phenotype reported for the p63−/− mice was not fully reverted. However, there was a greater degree of re-epithelialisation than in the selective ΔNp63<i>α</i> genetic complementation, and much greater than in the p63−/− and p63−/−;TA mice. These epidermal patches expressed K5 and K14 (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig6">Figure 6c</a>), which normally characterise basal layers, together with K1 and loricrin (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig6">Figure 6d</a>), which are usually associated with the upper layers. The skin patches in the p63−/−;ΔN;TA (mice knockout for p63, re-expressing both ΔNp63<i>α</i> and TAp73<i>α</i>) mice had a greater degree of organisation and were more differentiated (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig7">Figure 7</a>). Definite flattening and layering of the superficial cells was observed in many of these patches, but cornification was invariably incomplete and no corneodesmosomes or traces of intercellular lipid were found. Throughout the epidermis, keratin filaments were usually organised into fibrils and were associated with fibrillar granules in the more superficial layers (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig7">Figure 7</a>). Underlying some of the skin patches were traces of a basement membrane together with numerous but indistinct hemidesmosomes (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig7">Figure 7d</a>). The results indicate that reintroduction of TAp63<i>α</i> and ΔNp63<i>α</i> not only expanded the number of keratinocytes and consequently expanded the expression of epidermal-specific proteins, but the proteins expressed (filaggrin, keratins, loricrin) are assembled correctly in the epidermis (i.e. filaggrin granules associated with keratin filaments). More importantly, in the double-complemented mice, we detected basal lamina and hemidesmosomes, structures that are crucial for the formation of a functional pluristratified epithelium and which are absent in the null mice. This is of particular interest as p63 has been recently demonstrated to be essential for the symmetry of cell division.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 14" title="Lechler T and Fuchs E (2005) Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 437: 275–280." href="/articles/4401926#ref-CR14" id="ref-link-section-d42514748e1258">14</a></sup></p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-6" data-title="Figure 6"><figure><figcaption><b id="Fig6" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 6</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/6" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig6_HTML.jpg?as=webp"><img aria-describedby="Fig6" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig6_HTML.jpg" alt="figure 6" loading="lazy" width="685" height="533"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-6-desc"><p>Reintroduction of both TAp63<i>α</i> and ΔNp63<i>α</i> into the p63−/− mice. (<b>a</b>) Newborn p63−/−;ΔN;TA show significant degree of re-epithelialisation. (<b>b</b>) Histology (haematoxylin and eosin (H&E)). Areas of re-epithelialisation are evident. Bars=250 <i>μ</i>m (upper panel) or 50 <i>μ</i>m (lower panel). (<b>c</b>, <b>d</b>) Confocal immunostaining of skin biopsies of newborn mice. Reintroduction of both ΔNp63<i>α</i> and TAp63<i>α</i> into p63−/− mice allows a greater degree of re-epithelialisation, with expression of differentiated basal (K5, K14) and upper (K1, loricrin) layer markers. White stars indicate autofluorescent blood cells, also visible in unstained slides. Bar=50 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/6" data-track-dest="link:Figure6 Full size image" aria-label="Full size image figure 6" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-7" data-title="Figure 7"><figure><figcaption><b id="Fig7" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 7</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/7" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig7_HTML.jpg?as=webp"><img aria-describedby="Fig7" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig7_HTML.jpg" alt="figure 7" loading="lazy" width="413" height="461"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-7-desc"><p>Ultrastructure of epithelial patches following reintroduction of TAp63<i>α</i> and ΔNp63<i>α</i> in p63−/− mice by genetic complementation showing filaggrin granules (white arrows) and loricrin granules (black arrows). (<b>a</b>–<b>d</b>) TAp63<i>α</i> and ΔNp63<i>α</i> genetically complemented into p63−/− mice. (<b>a</b>) Partially cornified squamous cells predominate in the superficial layers and numerous keratin fibrils are present in most cells of the epithelium; the detail is reported as (<b>b</b>). (<b>b</b>) (detail of (<b>a</b>)) Keratin filaments are associated with filaggrin granules in the granular layer. (<b>c</b>) Epithelial cells (left) are separated from the underlying connective tissue by a basement membrane (curved arrows); the detail is reported in (<b>d</b>). (<b>d</b>) (detail of (<b>c</b>)). Numerous indistinct hemidesmosomes are also present at the base of the epithelial cells (black arrowheads). All bars=1 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/7" data-track-dest="link:Figure7 Full size image" aria-label="Full size image figure 7" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><h3 class="c-article__sub-heading" id="Sec5">Biochemical evaluation of epidermal proteins</h3><p>In order to address whether the primitive epidermis in these animals had become detached mechanically in the birth canal, we isolated embryos at E19.5 and washed extensively for 24 h in a buffer (1% SDS and 20 mM DTT) to solubilise the epidermal proteins; some degree of mechanical friction was also gently applied. This procedure may produce a more representative quantification of the epidermal proteins expressed in the different transgenic mice. K14, filaggrin and loricrin were expressed, although to a limited extent, in p63−/− mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig4">Figure 4a–c</a>), demonstrating that their production can be regulated by genes other than p63. Reintroduction of TAp63<i>α</i> did not significantly increase expression of these proteins. However, consistent with the previous data, selective complementation with ΔNp63<i>α</i> resulted in significantly enhanced expression of K14 and filaggrin, which was further enhanced in the double p63−/−;ΔN;TA complemented mice. Loricrin in normal skin is stored in L-granules (see electron microscopy results) before being crosslinked by transglutaminases (TG) in the upper granular layer, and can be solubilised by SDS buffer and detected mainly as a monomer in Western blot (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig4">Figure 4c</a>). In contrast, in the p63−/− and p63−/− complemented mice, which do not have proper granular layer formation, loricrin is not stored in the normal, small granules. In these animals, any loricrin in the abnormally large, osmiophilic granules may be sequestered from membrane-bound enzymes, but cytoplasmic loricrin is immediately crosslinked by TG. Indeed, the presence of crosslinked loricrin (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig4">Figure 4c</a>) reflects the expression and enzymatic activation of TG in non-complemented p63−/− mice, which is enhanced after transgenic complementation.</p><h3 class="c-article__sub-heading" id="Sec6">Molecular targets of the individual p63 isoforms</h3><p>As the <i>in vivo</i> evidence indicated that ΔNp63<i>α</i> facilitates the formation of the basal layer, we decided to investigate the underlying molecular mechanisms. As TP63 is a transcription factor, we performed a gene array analysis using Tet-On inducible cell lines expressing individual p63 isoforms.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Gressner O, Schilling T, Lorenz K, Schulze Schleithoff E, Koch A, Schulze-Bergkamen H, Maria Lena A, Candi E, Terrinoni A, Valeria Catani M, Oren M, Melino G, Krammer PH, Stremmel W and Muller M (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24: 2458–2471." href="/articles/4401926#ref-CR15" id="ref-link-section-d42514748e1415">15</a></sup></p><p>Several genes are regulated by TAp63<i>α</i> and ΔNp63<i>α</i>. A total of 171 genes are upregulated by TAp63<i>α</i>, of which 163 are unique for TAp63<i>α</i> and eight are in common with ΔNp63<i>α</i>. TAp63<i>α</i> downregulates 60 genes, 55 of which are unique, whereas five are in common with ΔNp63a. In contrast, ΔNp63<i>α</i> upregulates fewer genes (52), probably by using the second TA domain;<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 16" title="Ghioni P, Bolognese F, Duijf PH, Van Bokhoven H, Mantovani R and Guerrini L (2002) Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains. Mol. Cell. Biol. 22: 8659–8668." href="/articles/4401926#ref-CR16" id="ref-link-section-d42514748e1443">16</a></sup> among these, 44 are unique for ΔNp63<i>α</i> and eight are in common with TAp63<i>α</i>. ΔNp63<i>α</i> also downregulates 30 genes, 25 of which are unique and the other five are in common with TAp63<i>α</i> (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig8">Figure 8a and b</a>). TAp63<i>α</i> shows high transcriptional activity and, in particular, drives the expression of proteins specific to the upper layer of the epidermis, such as Ets-1, K1, K10, profilaggrin, involucrin and TG type 3 and 5. Conversely, ΔNp63<i>α</i> induced, with a high log ratio, the expression of genes present in the basal layer, such as K14. The results obtained in gene array analysis were confirmed using real-time PCR (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig8">Figure 8c</a>). Accordingly, we searched the promoter region of K14 gene for potential binding sites. As shown in <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">Figure 9a</a>, three p53-like responsive sites were identified located at −1813/−1833, −1368/−1388 and −114/−134, respectively. This promoter region was cloned in a plasmid upstream of a luciferase reporter gene. We observed that ΔNp63<i>α</i>, but not TAp63<i>α</i>, significantly increased luciferase activity in a dose-dependent manner (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">Figure 9b</a>). The ΔNp63<i>α</i>-dependent transactivation strictly depends on the p53-like responsive element III (RE III), as deletion of the first and second p53-like consensus sites (Δ1 and Δ2) has no effect on the ability of ΔNp63<i>α</i> to drive the K14 promoter, whereas deletion of the third site (Δ3) abolishes ΔNp63<i>α</i> transcriptional activity (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">Figure 9b</a>). In order to verify whether p63 was able to directly interact with these consensus sequences, we performed a chromatin immunoprecipitation assay (ChIP) using HaCat cells. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">Figure 9c</a> shows the ability of endogenous p63 protein to bind directly the third p53-like binding (RE III) <i>in vivo</i>. By Western blot, we confirmed that ΔNp63<i>α</i>, but not TAp63<i>α</i>, transfected in HEK293 epithelial cells induced the expression of the K14 protein (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">Figure 9d</a>). Together, these data indicate that ΔNp63<i>α</i> is able to drive, both <i>in vitro</i> and <i>in vivo</i>, the expression of basal layer markers of the epidermis, such as K14, whereas TAp63<i>α</i> is able to drive the expression of proteins of the upper layers of the epidermis.</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-8" data-title="Figure 8"><figure><figcaption><b id="Fig8" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 8</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/8" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig8_HTML.jpg?as=webp"><img aria-describedby="Fig8" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig8_HTML.jpg" alt="figure 8" loading="lazy" width="685" height="668"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-8-desc"><p>DNA microarray analysis of p63 isoforms. (<b>a</b>) Plot of genes regulated by TAp63<i>α</i> or ΔNp63<i>α</i> isoforms in Tet-inducible Saos-2 cells. The inducible cell lines are described by Gressner <i>et al.</i>,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Gressner O, Schilling T, Lorenz K, Schulze Schleithoff E, Koch A, Schulze-Bergkamen H, Maria Lena A, Candi E, Terrinoni A, Valeria Catani M, Oren M, Melino G, Krammer PH, Stremmel W and Muller M (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24: 2458–2471." href="/articles/4401926#ref-CR15" id="ref-link-section-d42514748e1548">15</a></sup> as well as the gene array. (<b>b</b>) Regulation of selected epidermal genes detected in the microarray analysis. The data presented are mean±S.D., <i>n</i>=3. (<b>c</b>) Validation of target genes, identified in (<b>b</b>), by quantitative real-time PCR. The panels show representative results of two independent inductions. A representative experiment is shown</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/8" data-track-dest="link:Figure8 Full size image" aria-label="Full size image figure 8" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-9" data-title="Figure 9"><figure><figcaption><b id="Fig9" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 9</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/9" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig9_HTML.jpg?as=webp"><img aria-describedby="Fig9" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig9_HTML.jpg" alt="figure 9" loading="lazy" width="685" height="288"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-9-desc"><p>ΔNp63<i>α</i> directly transactivates the K14 promoter. (<b>a</b>) Map of the human K14 promoter region. The grey boxes represent p53-like responsive elements; corresponding bps, sequences and restriction enzymes used to generate deleted constructs are indicated (Δ1–3). (<b>b</b>) ΔNp63<i>α</i> transactivates the K14 promoter in a dose-dependent manner, as indicated in the luc assay. Deletion of the third p53-like RE completely abrogates transactivation by ΔNp63<i>α</i>. The luc assay (shown) was performed in Saos-2 cells, and similar results were also obtained in HEK293 cells (not shown). Three independent experiments were performed, and a representative result is shown (mean±S.D., <i>n</i>=3). (<b>c</b>) ChIP of p63 protein on the K14 promoter using oligonucleotides for the p53-like binding sites 1–2 or 3. The ChIP was performed using nuclear extracts from HaCat cells (lane 1: marker; lane 2: non-specific antibody (nsp); lane 3: specific antibody anti-p63 (sp); lane 4: input. A representative result of two independent experiments is shown. (<b>d</b>) Western blot of K14 induced by ΔNp63<i>α</i> upon transient transfection in HEK293 cells. Cells were co-transfected with a plasmid containing GFP in order to monitor transfection efficiency. Western blot of actin was used as loading control. A representative result of two independent experiments is shown</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/9" data-track-dest="link:Figure9 Full size image" aria-label="Full size image figure 9" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div><p>The effect of the overexpression of p63 isoforms on the expression of K14, K1 and involucrin was then determined using the previously described TAp63<i>α</i> and ΔNp63<i>α</i> transgenic mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig10">Figure 10a and b</a>). K14 is upregulated in ΔNp63<i>α</i> but not in TAp63<i>α</i> transgenic mice. In contrast, involucrin and K1 are the only markers upregulated in TAp63<i>α</i> transgenic mice. Together, these results are in agreement with the data obtained from the gene array analysis (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig8">Figures 8</a> and <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">9</a>), and with the effect of the ΔNp63α <i>in vivo</i> genetic complementation in the p63-knockout mice (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig3">Figures 3</a> and <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig6">6</a>). All indicate a role for ΔNp63<i>α</i> in the formation of the basal layer of epidermis.</p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-10" data-title="Figure 10"><figure><figcaption><b id="Fig10" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 10</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/10" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig10_HTML.jpg?as=webp"><img aria-describedby="Fig10" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig10_HTML.jpg" alt="figure 10" loading="lazy" width="685" height="428"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-10-desc"><p><i>In vivo</i> validation, in transgenic mice overexpressing either TAp63<i>α</i> or ΔNp63<i>α</i> isoforms, of K14 as a target of ΔNp63<i>α</i>, identified in <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig8">Figures 8</a> and <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig9">9</a>. (<b>a</b>, <b>b</b>) Expression of epidermal proteins in transgenic mice overexpressing TAp63<i>α</i> or ΔNp63<i>α</i> isoforms under the control of the K5 promoter; see also <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig1">Figure 1</a>. (<b>a</b>) Confocal images of K14, K1 and involucrin in the epidermis of both newborn wt and transgenic mice. Bar=50 <i>μ</i>m. (<b>b</b>) Immunohistochemistry of K14 staining in different wt and transgenic newborn mice. Bar=100 <i>μ</i>m</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/10" data-track-dest="link:Figure10 Full size image" aria-label="Full size image figure 10" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div></div></div></section><section data-title="Discussion"><div class="c-article-section" id="Sec7-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec7">Discussion</h2><div class="c-article-section__content" id="Sec7-content"><p>Several studies have shown both a fundamental role for p63 in epithelial formation, and that p63 is upstream of many genes involved in epithelial development, although no distinct function for the isomeric forms encoded by the two promoters (ΔNp63, TAp63) has previously been established <i>in vivo</i>. Although the epidermal phenotype of the p63 null mice reported by two independent laboratories was similar, their interpretation of the underlying mechanisms was in sharp contrast. Whereas Roop and co-workers<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR and Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713." href="/articles/4401926#ref-CR10" id="ref-link-section-d42514748e1736">10</a></sup> concluded that p63 controls epidermal differentiation, McKeon and co-workers<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e1740">11</a></sup> suggested that epidermal differentiation was normal but that the defect lay in the proliferation potential of the stem cell compartment. In an attempt to resolve this issue, we have generated p63 null mice selectively expressing ΔNp63<i>α</i> or TAp63<i>α</i>, or both isoforms together. The data are consistent with a role for ΔNp63<i>α</i> in controlling expansion of progenitor cells in the basal layer, allowing TAp63<i>α</i> acting synergistically and/or subsequently to control differentiation of upper epidermal layers.</p><p>Although reversion of the gross p63 null phenotype was only partial even when both p63 isoforms were introduced, the transgenes were expressed under conditions that may not fully reflect their physiological temporal, spatial and quantitative expression. Moreover, complementation was only performed with the C-terminal <i>α</i> isomers, and an additional role for <i>β</i> and <i>γ</i> isomers cannot be excluded. Nevertheless, the degree of reconstitution of the gross morphological, histological and ultrastructural phenotype is greater when ΔNp63 is selectively reintroduced compared with complementation only with TAp63, although the structural recovery is even more pronounced by complementation with both isoforms. In addition, ΔNp63 transactivates genes, such as K14, which are characteristic of the basal layer, whereas transactivation of upper layer genes, such as K1, K10, filaggrin and loricrin, is mediated by TAp63. However, as expression of these upper epithelial proteins is also increased in mice selectively complemented with ΔNp63, other, non-p63 transactivators must also be able to contribute to their expression.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 17" title="De Laurenzi V, Rossi A, Terrinoni A, Barcaroli D, Levrero M, Costanzo A, Knight RA, Guerrieri P and Melino G (2000) p63 and p73 transactivate differentiation gene promoters in human keratinocytes. Biochem. Biophys. Res. Commun. 273: 342–346." href="/articles/4401926#ref-CR17" id="ref-link-section-d42514748e1769">17</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 18" title="Lapi E, Iovino A, Fontemaggi G, Soliera AR, Iacovelli S, Sacchi A, Rechavi G, Givol D, Blandino G and Strano S (2006) S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation. Oncogene [Epub ahead of print]." href="/articles/4401926#ref-CR18" id="ref-link-section-d42514748e1772">18</a></sup> The data are also consistent with reports that overexpression of ΔNp63 in primary murine keratinocytes abrogates calcium-induced growth arrest and blocks expression of maturation-specific proteins, such as K10 and filaggrin.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 19" title="King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF and Weinberg WC (2003) deltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 22: 3635–3644." href="/articles/4401926#ref-CR19" id="ref-link-section-d42514748e1776">19</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 20" title="King KE, Ponnamperuma RM, Gerdes MJ, Tokino T, Yamashita T, Baker CC and Weinberg WC (2006) Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis. Carcinogenesis 27: 53–63." href="/articles/4401926#ref-CR20" id="ref-link-section-d42514748e1779">20</a></sup></p><p>Therefore, within the limitations of our experimental model, our data support a crucial role for p63 in epidermal development (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/4401926#Fig11">Figure 11</a>). The evidence presented here that ΔNp63 is more important for maintaining the proliferative potential of the basal layer, whereas TAp63, according to others,<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 21" title="Ihrie RA, Marques MR, Nguyen BT, Horner JS, Papazoglu C, Bronson RT, Mills AA and Attardi LD (2005) Perp is a p63-regulated gene essential for epithelial integrity. Cell 120: 843–856." href="/articles/4401926#ref-CR21" id="ref-link-section-d42514748e1788">21</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 22" title="Koster MI, Kim S, Mills AA, DeMayo FJ and Roop DR (2004) p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev. 18: 126–131." href="/articles/4401926#ref-CR22" id="ref-link-section-d42514748e1791">22</a></sup> contributes to the formation of the upper stratified layers suggests that the two isoforms act in concert, and helps to resolve the previously conflicting interpretations of the role of p63 in the development and maintenance of epithelia.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 23" title="McKeon F (2004) p63 and the epithelial stem cell: more than status quo? Genes Dev. 18: 465–469." href="/articles/4401926#ref-CR23" id="ref-link-section-d42514748e1795">23</a>, <a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 24" title="Nicotera P and Melino G (2005) Neurodevelopment on route p63. Neuron 48: 707–709." href="/articles/4401926#ref-CR24" id="ref-link-section-d42514748e1798">24</a></sup></p><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-11" data-title="Figure 11"><figure><figcaption><b id="Fig11" class="c-article-section__figure-caption" data-test="figure-caption-text">Figure 11</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><a class="c-article-section__figure-link" data-test="img-link" data-track="click" data-track-label="image" data-track-action="view figure" href="/articles/4401926/figures/11" rel="nofollow"><picture><source type="image/webp" srcset="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig11_HTML.jpg?as=webp"><img aria-describedby="Fig11" src="//media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fsj.cdd.4401926/MediaObjects/41418_2006_Article_BF4401926_Fig11_HTML.jpg" alt="figure 11" loading="lazy" width="407" height="417"></picture></a></div><div class="c-article-section__figure-description" data-test="bottom-caption" id="figure-11-desc"><p>Proposed scheme of action of the p63 isoforms deducted from the experiment reported. Within the limit of our experimental model, the data reported indicate that (i) p63−/− epidermis shows very limited and only partially differentiated epithelial areas, indicating that p63 is not strictly required for the expression of epithelial differentiation proteins (as loricrin, involucrin and K1/K10 are expressed in p63 null animals); our data are consistent with p63−/− mice retaining a limited expansion of the proliferative compartment (patchy epidermis), which in places is able to differentiate (expression of loricrin, involucrin, K1/K10); (ii) ΔNp63<i>α</i> significantly increases the number of epidermal basal cells, consequently allowing a greater degree of differentiation, and therefore ΔNp63 would appear to be the isoform responsible for the proliferation potential function (expansion of the basal layer); (iii) both TAp63<i>α</i> and ΔNp63<i>α</i> are required for epidermal development, as single genetic complementation does not fully revert the null phenotype; and therefore, (iv) TAp63<i>α</i> and ΔNp63<i>α</i> have different, complementary, synergistic functions necessary for the complete formation of the epidermis, where TAp63 facilitates the expression of differentiation markers (but is not strictly required, as p63−/− also express these proteins to a limited extent; see point (i)); (v) p63 exerts its effects at least in part by acting directly upstream of K14 (directly transactivated by ΔNp63<i>α</i>) and Ets-1, K1, TG and involucrin (inv) (directly transactivated by TAp63<i>α</i>)</p></div></div><div class="u-text-right u-hide-print"><a class="c-article__pill-button" data-test="article-link" data-track="click" data-track-label="button" data-track-action="view figure" href="/articles/4401926/figures/11" data-track-dest="link:Figure11 Full size image" aria-label="Full size image figure 11" rel="nofollow"><span>Full size image</span><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-right-small"></use></svg></a></div></figure></div></div></div></section><section data-title="Materials and Methods"><div class="c-article-section" id="Sec8-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec8">Materials and Methods</h2><div class="c-article-section__content" id="Sec8-content"><h3 class="c-article__sub-heading" id="Sec9">Primary keratinocyte, cell cultures and imaging</h3><p>Primary keratinocytes were isolated according to Yuspa <i>et al.</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 25" title="Yuspa SH, Kilkenny AE, Steinert PM and Roop DR (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J. Cell Biol. 109: 1207–1217." href="/articles/4401926#ref-CR25" id="ref-link-section-d42514748e1855">25</a></sup> from the skin of E19.5 neonates. The skin was floated overnight on trypsin/EDTA at 4°C and primary keratinocytes were isolated. Cells were cultured on collagen-coated dishes in medium supplemented with 0.05 mM Ca<sup>2+</sup> and 5 ng/ml epidermal growth factor. TAp63<i>α</i>- and ΔNp63<i>α</i>-inducible Saos-2 cells were as described by Gressner <i>et al.</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Gressner O, Schilling T, Lorenz K, Schulze Schleithoff E, Koch A, Schulze-Bergkamen H, Maria Lena A, Candi E, Terrinoni A, Valeria Catani M, Oren M, Melino G, Krammer PH, Stremmel W and Muller M (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24: 2458–2471." href="/articles/4401926#ref-CR15" id="ref-link-section-d42514748e1870">15</a></sup> Embryos or tissues were fixed in 4% paraformaldehyde; microwave-assisted antigen retrieval was performed in 0.01 M sodium citrate (pH 6) for three cycles of 5 min followed by cooling at 50°C. Sections were incubated overnight with primary and secondary antibodies for confocal microscopy (Nikon, Tokyo, Japan, C1). Samples for electron microscopy were fixed in formaldehyde/glutaraldehyde and osmium tetroxide before being embedded in epoxy resin. Sections (1 <i>μ</i>m) were stained with toluidine blue and examined by light microscopy, to select areas for electron microscopy. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate before examination (Zeiss, Gottingen, Germany, 902A); see below.</p><h3 class="c-article__sub-heading" id="Sec10">Transgenic mice</h3><p>A recombination vector containing the K5 promoter (from Dr. Manfred Blessing, Joannes Gutemberg University, Mainz, Germany) and the PolyA+ signal was used.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Breuhahn K, Mann A, Muller G, Wilhelmi A, Schirmacher P, Enk A and Blessing M (2000) Epidermal overexpression of granulocyte–macrophage colony-stimulating factor induces both keratinocyte proliferation and apoptosis. Cell Growth Differ. 11: 111–121." href="/articles/4401926#ref-CR26" id="ref-link-section-d42514748e1885">26</a></sup> Mouse TAp63<i>α</i> and ΔNp63<i>α</i> were tagged at the N-terminus (HA) and cloned into the vector using <i>Cla</i>I restriction sites located between the promoter and the polyA+ cassette. The fragment containing the expression cassette (K5 promoter/TAp63<i>α</i>-ΔNp63<i>α</i>/polyA+ signal) was released using <i>Kpn</i>I. The transgenic mice generated are on a pure C57/BL6 background. The p63+/− mice are on a mixed background (129svJ, C57BL/6, see Yang <i>et al.</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 11" title="Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718." href="/articles/4401926#ref-CR11" id="ref-link-section-d42514748e1910">11</a></sup>).</p><p>The genotype was determined from extracted DNA (Qiagen, Valencia, California, USA; see the manufacturer's instructions). PCR was as follows: 95°C × 3 min and 30 s, 40 × (94°C × 30 s, 50°C × 35 s, 72°C × 45 s), 72°C × 5 min using as forward primer ATA CGA TGT TCC AGA TTA GG and reverse primer TGT TCA TTC CTC CGA CGC AGC. The conditions for PCR screening of p63 +/− mice were as follows: 95°C × 3 min and 30 s, 40 × (94°C × 30 s, 60°C × 30 s, 72°C × 1 min and 30 s), 72°C × 5 min using as forward primer TTC TCA GAT GGT ACC GCT CC and reverse primers GGT GCT TTG AGG CCC GGA TC and GAA AGC GAA GGA GCA AAG CTG.</p><h3 class="c-article__sub-heading" id="Sec11">Confocal imaging</h3><p>Embryos or tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax and cut. After wax removal (Histolemon, Carlo Erba, Italy), sections were rehydrated in alcohol/distilled water. Microwave-assisted antigen retrieval was performed in 0.01 M sodium citrate (pH 6) for three cycles of 5 min (300 W) followed by cooling at 50°C and a final round of microwaving for 5 min, cooling and a rinse in PBS. Nonspecific antigens were blocked by incubation in 10% goat serum in PBS for 2 h at 4°C. Sections were incubated overnight with the following primary antibodies: monoclonal anti-p63 (Ab4, Neomarkers, Fremont, California, USA; 1/500 dilution), polyclonal anti-MK14 (PRB-155P, Covance; 1/3000 dilution), polyclonal anti-MK5 (PRB-160P, Covance, 1/500 dilution), polyclonal anti-MK1 (PRB-165P, Covance; 1/500 dilution), polyclonal anti-involucrin (PRB-142C, Covance; 1/500 dilution), polyclonal anti-filaggrin (PRB-417P, Covance; Princetown, NY, USA; 1/500 dilution), anti-TGase3 (1 : 200; from R Schmidt, L'Oreal, Paris), polyclonal anti-loricrin (1 : 100),<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 10" title="Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR and Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713." href="/articles/4401926#ref-CR10" id="ref-link-section-d42514748e1937">10</a></sup> Troma-1 (polyclonal anti-K8; 1 : 10 000 dilution; from Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Sections were washed three times with PBS and incubated for 1 h with the secondary antibodies conjugated with FITC or PE. Following two washes in PBS, the tissue sections were incubated for 5 min with DAPI to reveal the nuclei. The tissue sections were then mounted using the Prolong Antifade kit. Fluorescence was evaluated by confocal microscopy (Nikon, C1 on Eclipse TE200; EZC1 software) fitted with an argon laser (488 nm excitation), a He/Ne laser (542 nm excitation) and UV excitation at 405 nm (DAPI staining) from a blue diode.</p><h3 class="c-article__sub-heading" id="Sec12">Electron microscopy</h3><p>Samples were fixed in formaldehyde/glutaraldehyde and osmium tetroxide before being embedded in epoxy resin. Sections (1 <i>μ</i>m) were stained with toluidine blue and examined by light microscopy, to select areas for electron microscopy. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate before examination in a Zeiss 902A electron microscope. Several ultrathin sections were treated with 10% hydrogen peroxide, before staining, to confirm the composition of loricrin granules.<sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 27" title="Jessen H (1970) Two types of keratohyalin granules. J. Ultrastruct. Res. 33: 95–115." href="/articles/4401926#ref-CR27" id="ref-link-section-d42514748e1952">27</a></sup></p><h3 class="c-article__sub-heading" id="Sec13">Real-time PCR assay</h3><p>RNA was extracted from control and doxycycline-induced Saos-2 TetOn-TAp63 and Saos-2 TetOn-ΔNp63 cells, using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). A 1 <i>μ</i>g portion of RNA was used for reverse transcription using the InPromII kit (Promega, Madison, WI, USA) and 1/5 of the reaction used for PCR. Real-time PCR was performed using the Platinum SYBR Green qPCR SuperMix UDG with Rox (Invitrogen; cat. no. 11744-500), with an amplification programme as follows: one cycle of 95°C for 3 min and 40 cycles of 94°C for 15 s and 59°C for 50 s. The reaction was followed by a melting curve protocol according to the specification of the ABI 7000 instrument (Applied Biosystem, Foster City, CA, USA). Amplicon sizes were between 200 and 230 bp. The primers used were as follows: TGM5 (+AGC CTG CAT ACA CCT TCC CTT C; −AAC GCT GTG TCC TGC CAG AAT G), TGM3 (+TAT CAG CAT CTC CAG TCC TGC C; −GCC AAT TCG GTT TGT GCT TCC), K1 (+TCA TCA ACT ACC AGC GCA GG; −ACC ATA ACC ACC ACC AAA GC), involucrin (+CAG GTC CAA GAC ATT CAA CC; −CAA GTT CAC AGA TGA GAC GG), filaggrin (+CAA TCA GGC ACT CAT CAC AC; −ACT GTT AGT GAC CTG ACT ACC), IKK-<i>α</i> (+GAA AAG GCC ATC CAC TAT GC; −TCA CCA TCT CTG TGC TGT C), p21 (+TGA GCG ATG GAA CTT CGA C; −ACA AGA CAG TGA CAG GTC C), ETS-1 (+TCA AGC CGA CTC TCA CCA TC; −CGA ACA TGG GTT TCT GTC CAC, hETS1R), <i>β</i>-actin (+AAA GAC CTG TAC GCC AAC A; −CGG AGT ACT TGC GCT CAG). <i>β</i>-Actin was used as a housekeeping gene for quantity normalisation. Relative quantisation of gene expression was performed according to the method described in ABI User Bulletin # 2 (updated October 2001).</p><h3 class="c-article__sub-heading" id="Sec14">Western blots and microarray</h3><p>Subconfluent HEK293 cells in 10 cm dishes were transfected with 15 <i>μ</i>g of total DNA using 30 <i>μ</i>l of lipofectamine 2000 reagent (Invitrogen). The amounts of HA-tagged TAp63<i>α</i> and ΔNp63<i>α</i> were normalised for protein level by transfecting 5 <i>μ</i>g of TAp63<i>α</i> vector and 15 <i>μ</i>g of ΔNp63<i>α</i> vector. Cells were harvested 24 h after transfection and lysed in RIPA buffer with protease inhibitors (Sigma, St. Louis, MI, USA) and 1 mM DTT (Sigma). Primary mouse keratinocytes were lysed in a buffer containing 100 mM NaCl, 1% Triton X-100, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 1 mM DTT. To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 <i>μ</i>g of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal anti-HA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution). Normalisation was achieved with a polyclonal anti-tubulin (H-235, Santa Cruz, 1 : 1000 dilution) or with a goat anti-actin antibody (C-11, Santa Cruz, 1 : 1000 dilution). After three washes in PBS-tween 20 (0.05%), HRP-conjugated secondary antibodies (Bio-Rad, goat anti-mouse, 170-5047 and goat anti-rabbit, 170-5046; and Santa Cruz, bovine anti-goat, sc-2384) were added (1 : 10 000–20 000 dilution in blocking solution). Proteins were detected using the ECL method.</p><p>TAp63<i>α</i>/ΔNp63<i>α</i> dox-inducible Saos-2 cells were as described by Gressner <i>et al.</i><sup><a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 15" title="Gressner O, Schilling T, Lorenz K, Schulze Schleithoff E, Koch A, Schulze-Bergkamen H, Maria Lena A, Candi E, Terrinoni A, Valeria Catani M, Oren M, Melino G, Krammer PH, Stremmel W and Muller M (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24: 2458–2471." href="/articles/4401926#ref-CR15" id="ref-link-section-d42514748e2080">15</a></sup> cRNAs were hybridised to the Genechip HuGene FL array (Affymetrix, Santa Clara, CA, USA) that contains probes for about 11 000 mRNA species. Scanned output files were inspected for hybridisation artefacts and further analysed using Genechip 3.3 software (Affymetrix). Ratios were obtained by dividing the average difference of TAp63<i>α</i> and ΔNp63 for each time point with those of the 0 h time point.</p><h3 class="c-article__sub-heading" id="Sec15">Chromatin immunoprecipitation and luciferase assay</h3><p>Cells (1.5 × 10<sup>6</sup>) were crosslinked for 5 min, with 1% formaldehyde, 5 mM Hepes-KOH pH 8.0, 0.1 mM EDTA and 10 mM NaCl. Crosslinking was stopped by incubating cells with 0.125 M glycine for 5 min at RT. After washing with ice-cold PBS, cells were harvested in lysis buffer (50 mM Hepes-KOH pH 8.0, 1 mM EDTA, 140 mM NaCl, 25% glycerol, 0.5% NP-40, 0.25% Triton X-100) plus protease inhibitors. The nuclei were collected by centrifugation and resuspended in wash buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl) plus protease inhibitors. After centrifugation, the nuclei were resuspended in 1.8 ml ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 0.0167 M Tris-HCl, 0.167 M NaCl) plus 200 <i>μ</i>l SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1). Cell lysates were sonicated in order to obtain chromatin fragments of <span class="stix">∼</span>700 bp. After centrifugation at 13 000 r.p.m. for 20 min to remove any cell debris, 100 <i>μ</i>g of total proteins was precleared with 100 <i>μ</i>l of protein A-agarose/salmon sperm DNA (Upstate, Charlottesville, VA, USA) and 2 <i>μ</i>g of mouse IgG<sub>1</sub><i>κ</i> (BD, Franklin Lakes, NJ, USA) for 2 h at 4°C. Then, the precleared extracts were incubated with 2 <i>μ</i>g monoclonal anti-HA (16B12, Covance) or monoclonal anti-p63 (Ab4, Neomarkers) overnight at 4°C, followed by incubation with protein A-agarose/salmon sperm DNA (60 <i>μ</i>l) for 1 h 30 min at 4°C. The negative control was incubated with 2 <i>μ</i>g mouse anti-K5 (Santa Cruz). The immune complexes were washed twice with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 0.15 M NaCl), five times with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 0.5 M NaCl), once with LiCl salt wash buffer (1 mM EDTA, 10 mM Tris-HCl, 0.25 M LiCl, 1% NP-40, 1% deoxycholate) and twice with TE buffer. The precipitates were extracted twice using 250 <i>μ</i>l of IP elution buffer (1% SDS, 0.1 M NaHCO3). The total eluates (500 <i>μ</i>l) were pooled by adding 20 <i>μ</i>l of 5 M NaCl and incubated at 65°C for at least 6 h to reverse the formaldehyde crosslinking. DNA fragments were purified by phenol–chloroform extraction and ethanol precipitation, and dissolved in 30 <i>μ</i>l of sterile water.</p><p>DNA samples were then analysed with 38 cycles of PCR to amplify K14 promoter sequences (94°C for 30 s, 58°C for 40 s, 72°C for 40 s). We used a primer pair amplifying the first and second p53 responsive element in the K14 promoter (forward 5′-CCTCTTCGGCCGGTGGAC-3′ and reverse 5′-CCGTTTTCGACCCTGAGAG-3′) and two other primers amplifying the third p53 responsive element (forward 5′-CCTCTTCGGCCGGTGGAC-3′ and reverse 5′-CCGTTTTCGACCCTGAGAG-3′).</p><p>Subconfluent HEK293 cells in 12-well dishes were transfected with Effectene-Liposomal Transfection Reagent (Qiagen, Hilden, Germany) at a 1 : 3 ratio between the reporter plasmid (containing the firefly luciferase gene under the control of K14 promoter) and the expression vectors encoding for TAp63<i>α</i> and ΔNp63<i>α</i>. When needed, the pcDNA empty vector (Invitrogen) was added to reach the total amount of DNA (400 ng) used in each transfection. In all cases, 10 ng of <i>Renilla</i> Luciferase Vector (pRL-CMV; Promega, Madison, WI, USA) was co-transfected, as a control of transfection efficiency. At 24 h after transfection, luciferase activities of cellular extracts were measured, by using a Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA); light emission was measured over 10 s using an OPTOCOMP I luminometer. Efficiency of transfection was normalised using <i>Renilla</i> luciferase activity.</p></div></div></section> </div> <div class="u-mt-32"> <section data-title="Abbreviations"><div class="c-article-section" id="abbreviations-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="abbreviations">Abbreviations</h2><div class="c-article-section__content" id="abbreviations-content"><dl class="c-abbreviation_list"><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>TA:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>transactivation domain</p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>ΔN:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>amino-terminal truncated protein</p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>H&E:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>haematoxylin and eosin</p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>tg:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>transgenic mice</p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>p63−/−;ΔN:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>mice knockout for p63, re-expressing ΔNp63<i>α</i></p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>p63−/−;TA:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>mice knockout for p63, re-expressing TAp63<i>α</i></p> </dd><dt class="c-abbreviation_list__term u-text-bold u-float-left u-pr-16" style="min-width:50px;"><dfn>p63−/−;ΔN;TA:</dfn></dt><dd class="c-abbreviation_list__description u-mb-24"> <p>mice knockout for p63, re-expressing both ΔNp63<i>α</i> and TAp73<i>α</i></p> </dd></dl></div></div></section><div id="MagazineFulltextArticleBodySuffix"><section aria-labelledby="Bib1" data-title="References"><div class="c-article-section" id="Bib1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Bib1">References</h2><div class="c-article-section__content" id="Bib1-content"><div data-container-section="references"><ol class="c-article-references" data-track-component="outbound reference" data-track-context="references section"><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="1"><p class="c-article-references__text" id="ref-CR1">Candi E, Schmidt R and Melino G (2005) The cornified envelope: a model of cell death in the skin. <i>Nat. 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This work was supported by grants from Telethon to EC; AIRC to VDL; and EU, AIRC, Telethon, FIRB, MIUR, MinSan, TelethonGGP04110 and MRC to GM.</p></div></div></section><section aria-labelledby="author-information" data-title="Author information"><div class="c-article-section" id="author-information-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="author-information">Author information</h2><div class="c-article-section__content" id="author-information-content"><h3 class="c-article__sub-heading" id="affiliations">Authors and Affiliations</h3><ol class="c-article-author-affiliation__list"><li id="Aff1"><p class="c-article-author-affiliation__address">Biochemistry Laboratory, IDI-IRCCS, c/o University of Rome ‘Tor Vergata’, Rome, 00133, Italy</p><p class="c-article-author-affiliation__authors-list">E Candi, A Rufini, A Terrinoni, M Ranalli, A Paradisi, L G Spagnoli, M V Catani, S Ramadan & G Melino</p></li><li id="Aff2"><p class="c-article-author-affiliation__address">Medical Research Council, Toxicology Unit, Leicester University, Leicester, LE1 9HN, UK</p><p class="c-article-author-affiliation__authors-list">D Dinsdale, V De Laurenzi, R A Knight & G Melino</p></li></ol><div class="u-js-hide u-hide-print" data-test="author-info"><span class="c-article__sub-heading">Authors</span><ol class="c-article-authors-search u-list-reset"><li id="auth-E-Candi-Aff1"><span class="c-article-authors-search__title u-h3 js-search-name">E Candi</span><div class="c-article-authors-search__list"><div class="c-article-authors-search__item c-article-authors-search__list-item--left"><a href="/search?author=E%20Candi" class="c-article-button" data-track="click" data-track-action="author link - 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Development 133: in press. Published online on March 8, 2006. doi: 10.1242/dev.0235) demonstrated that ΔNp63 is the main isoform expressed at all embryonic stages during epidermal, tooth and hair development, accounting for 100% of all p63 expressed up to E9 and 99% at E13. TAp63 expression starts at E13, and only accounts for 1% of total p63 protein expressed at this time. Thus, expression of the ΔNp63 isoform is predominant over that of TAp63, at a time before any epidermal stratification occurs. These results obtained by Laurikkala <i>et al.</i> are fully in agreement with our studies, using a completely different experimental approach, and confirm that the ΔNp63 isoform is crucial for the formation of the epidermis, whereas TAp63 contributes to the control of epithelial differentiation by acting synergistically and/or subsequently to ΔNp63.</p><p>Moreover, Laurikkala <i>et al.</i> also demonstrated that <i>Bmp 7, Fgfr2b, Jag1</i> and <i>Notch1</i> transcripts are coexpressed with ΔNp63 and are absent in p63−/−mice. Additional support for this complex pathway of regulations also comes from a further independent paper in press (Nguyen BC, Lefort K, Mandinova A, Antonini D, Devgan V, Della Gatta G, Koster MI, Zhang Z, Wang J, Tommasi di Vignano A, Kitajewski J, Chiorino G, Roop DR, Missero C and Dotto GP (2006). Cross- regulation between Notch and p63 in keratinocyte commitment to differentiation. Genes Dev. in press. doi:10.1101/gad1406006).</p></div></div></section><section data-title="Rights and permissions"><div class="c-article-section" id="rightslink-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="rightslink">Rights and permissions</h2><div class="c-article-section__content" id="rightslink-content"><p class="c-article-rights"><a data-track="click" data-track-action="view rights and permissions" data-track-label="link" href="https://s100.copyright.com/AppDispatchServlet?title=Differential%20roles%20of%20p63%20isoforms%20in%20epidermal%20development%3A%20selective%20genetic%20complementation%20in%20p63%20null%20mice&author=E%20Candi%20et%20al&contentID=10.1038%2Fsj.cdd.4401926&copyright=Springer%20Nature%20Limited&publication=1350-9047&publicationDate=2006-04-07&publisherName=SpringerNature&orderBeanReset=true">Reprints and permissions</a></p></div></div></section><section aria-labelledby="article-info" 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