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p63 in Skin Development and Ectodermal Dysplasias

  • Maranke I. Koster
    Correspondence
    Department of Dermatology, Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, Mail Stop 8320, Aurora, Colorado 80045, USA
    Affiliations
    Department of Dermatology, Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, Aurora, Colorado, USA
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      The transcription factor p63 is critically important for skin development and maintenance. Processes that require p63 include epidermal lineage commitment, epidermal differentiation, cell adhesion, and basement membrane formation. Not surprisingly, alterations in the p63 pathway underlie a subset of ectodermal dysplasias, developmental syndromes in which the skin and skin appendages do not develop normally. This review summarizes the current understanding of the role of p63 in normal development and ectodermal dysplasias.

      Abbreviations

      AEC
      ankyloblepharon ectodermal dysplasia and clefting
      EEC
      ectrodactyly, ectodermal dysplasia, and cleft lip
      EEM
      macular dystrophy
      SHFM
      split hand/foot malformation
      TA cells
      transit amplifying cells
      TDO
      tricho-dento-osseous
      SKPs
      skin-derived precursors

      Introduction

      The epidermis, the outermost component of the skin, functions as the primary barrier between the organism and the environment. It protects the organism from microbial, physical, and chemical assaults, as well as from excessive water loss. The barrier function of the epidermis is established during embryogenesis and is maintained postnatally by stem cells, which are located in the basal layer of the interfollicular epidermis (
      • Fuchs E.
      Finding one's niche in the skin.
      ). When interfollicular stem cells divide, they give rise to daughter cells, termed transit amplifying (TA) cells. After a few rounds of cell division, TA cells permanently withdraw from the cell cycle, and move suprabasally to initiate terminal differentiation (
      • Koster M.I.
      • Roop D.R.
      Mechanisms regulating epithelial stratification.
      ). This initial stage of terminal differentiation results in the formation of the spinous layer, the first suprabasal cell layer of the epidermis. Spinous keratinocytes subsequently differentiate into granular keratinocytes. Granular keratinocytes ultimately die when they form the stratum corneum, the outermost dead layer of the epidermis. The cells of the stratum corneum, termed corneocytes, replace their plasma membrane with a shell of cross-linked proteins. Together with extracellular lipids, these cross-linked proteins form the cornified cell envelope, which is the most important component of the epidermal water barrier (
      • Rice R.H.
      • Green H.
      The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein.
      ;
      • Steven A.C.
      • Steinert P.M.
      Protein composition of cornified cell envelopes of epidermal keratinocytes.
      ). As described in more detail below, the transcription factor p63 is required for key events in epidermal development and differentiation, including epidermal lineage commitment, keratinocyte adhesion, basement membrane formation, epidermal differentiation, and barrier formation. Thus, it comes as no surprise that alterations in the p63 pathway lead to developmental disorders in which these processes are affected.

      p63 in Developmental Disorders

      Developmental disorders caused by alterations in the p63 pathway

      As predicted from its critical function in the epidermis, abnormalities in the p63 pathway have been linked to several developmental disorders (Figure 1). First, p63 is mutated in several ectodermal dysplasias, a large group of developmental disorders in which ectodermal derivatives, such as the epidermis and its appendages, fail to develop normally (
      • Rinne T.
      • Brunner H.G.
      • van Bokhoven H.
      p63-associated disorders.
      ). Mutations in p63 were found to underlie several of these ectodermal dysplasias, including ectrodactyly, ectodermal dysplasia, and cleft lip (EEC) (
      • Celli J.
      • Duijf P.
      • Hamel B.C.
      • et al.
      Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.
      ) and ankyloblepharon ectodermal dysplasia and clefting (AEC or Hay–Wells syndrome) (
      • McGrath J.A.
      • Duijf P.H.
      • Doetsch V.
      • et al.
      Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63.
      ). Interestingly, even though both of these disorders are caused by mutations in p63, the patients have distinct abnormalities. For example, severe skin erosions are common in AEC patients, whereas they are rare in EEC patients (
      • Brunner H.G.
      • Hamel B.C.J.
      • van Bokhoven H.
      The p63 gene in EEC and other syndromes.
      ;
      • Julapalli M.R.
      • Scher R.K.
      • Sybert V.P.
      • et al.
      Dermatologic findings of ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome.
      ). Conversely, limb abnormalities in EEC patients are generally severe, whereas they are relatively mild in AEC patients (
      • Brunner H.G.
      • Hamel B.C.J.
      • van Bokhoven H.
      The p63 gene in EEC and other syndromes.
      ;
      • Bree A.F.
      Clinical lessons learned from the International Research Symposium on Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome.
      ;
      • Sutton V.R.
      • Plunkett K.
      • Dang D.X.
      • et al.
      Craniofacial and anthropometric phenotype in ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (Hay-Wells syndrome) in a cohort of 17 patients.
      ). These differences in phenotypic outcome are a reflection of the type of mutations found in EEC and AEC patients. Specifically, mutations in different functional domains of the p63 protein lead to different cellular defects, and thus to different diseases. In order to understand the etiology of these different but related diseases, we need to understand the functions of the different p63 proteins and protein domains.
      Figure thumbnail gr1
      Figure 1p63 in developmental disorders. Mutations in p63 or p63 target genes cause developmental disorders. Dominant mutations in p63 underlie ankyloblepharon ectodermal dysplasia and clefting (AEC), ectrodactyly, ectodermal dysplasia, and cleft lip (EEC), and split hand/foot malformation (SHFM)4, whereas dominant mutations in the p63 target genes P-cadherin, Dlx3, and Dlx5 and Dlx6 underlie ectodermal dysplasia, ectrodactyly, and macular dystrophy (EEM), tricho-dento-osseous (TDO) syndrome, and SHFM1, respectively. The common features of EEC, EEM, and SHFM include severe limb abnormalities, including syndactyly and ectrodactyly (right image; image depicts an EEC patient). A characteristic feature of AEC patients is the presence of severe erosions, often located to the scalp (left image). Other ectodermal dysplasias caused by mutations in p63 include limb-mammary syndrome and acro-dermato-ungual-lacrimal-tooth syndrome. Images of AEC and EEC patients are provided by the National Foundation for Ectodermal Dysplasias.
      By encoding two different N-termini (TA and ΔN) and multiple C-termini (α, β, γ, δ, and ε), the p63 gene generates multiple protein isoforms (
      • Yang A.
      • Kaghad M.
      • Wang Y.
      • et al.
      p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.
      ;
      • Mangiulli M.
      • Valletti A.
      • Caratozzolo M.F.
      • et al.
      Identification and functional characterization of two new transcriptional variants of the human p63 gene.
      ). All p63 isoforms contain identical DNA binding and oligomerization domains. Whereas mutations in the ΔN N-terminus or the α C-terminus cause AEC, mutations in the DNA binding domain cause EEC (Figure 2) (
      • Celli J.
      • Duijf P.
      • Hamel B.C.
      • et al.
      Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.
      ;
      • McGrath J.A.
      • Duijf P.H.
      • Doetsch V.
      • et al.
      Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63.
      ;
      • Rinne T.
      • Clements S.E.
      • Lamme E.
      • et al.
      A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp-Hodgkin/Hay-Wells-like syndromes.
      ). Nevertheless, the precise molecular pathway alterations that are triggered by these mutations are not yet understood.
      Figure thumbnail gr2
      Figure 2Mutations in different domains of p63 cause different ectodermal dysplasias. The p63 gene is expressed as multiple isoforms. Isoforms depicted here are the full-length isoform, TAp63α, and the predominantly expressed p63 isoform in late embryonic and postnatal epidermis, ΔNp63α. Mutations in the DNA-binding domain, common to all p63 isoforms, can cause ectrodactyly, ectodermal dysplasia, and cleft lip (EEC) or split hand/foot malformation (SHFM)4. Mutations in the ΔN N terminus or the SAM domain (only present in the α C terminus) underlie ankyloblepharon, ectodermal dysplasia, and clefting (AEC). For an overview of p63 mutations found in ectodermal dysplasia patients, refer to
      • Rinne T.
      • Brunner H.G.
      • van Bokhoven H.
      p63-associated disorders.
      ,
      • Rinne T.
      • Clements S.E.
      • Lamme E.
      • et al.
      A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp-Hodgkin/Hay-Wells-like syndromes.
      ). oligo, oligomerization domain; SAM, sterile alpha motif; TAD, transactivation domain.
      Another developmental disorder caused by p63 mutations is split hand/foot malformation (SHFM) (
      • Ianakiev P.
      • Kilpatrick M.W.
      • Toudjarska I.
      • et al.
      Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27.
      ). SHFM is a non-syndromic developmental disorder in which patients have severe limb abnormalities (ectrodactyly and syndactyly) but no other developmental defects (
      • Duijf P.H.G.
      • van Bokhoven H.
      • Brunner H.G.
      Pathogenesis of split-hand/split-foot malformation.
      ). Five independent chromosomal loci have been associated with SHFM (SHFM1–SHFM5). p63 was mapped to one of these loci, SHFM4, and mutations in p63 were found to underlie SHFM associated with this locus (Figures 1 and 2). Even though causative mutations have not been identified in the remaining four loci, candidate genes for SHFM have been mapped to these loci. For example, the SHFM1 locus harbors the DLX5 and DLX6 genes (
      • Scherer S.
      • Poorka] P.
      • Massa H.
      • et al.
      Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly.
      ). Interestingly, the limb phenotype of mice that lack both Dlx5 and Dlx6 phenocopies that of SHFM patients (
      • Merlo G.R.
      • Paleari L.
      • Mantero S.
      • et al.
      Mouse model of split hand/foot malformation type I.
      ;
      • Robledo R.F.
      • Rajan L.
      • Li X.
      • et al.
      The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development.
      ). Further, p63 directly induces Dlx5 and Dlx6 expression, suggesting that SHFM1 and SHFM4 are caused by alterations in the same genetic pathway (
      • Lo Iacono N.
      • Mantero S.
      • Chiarelli A.
      • et al.
      Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects.
      ).
      In addition to Dlx5 and Dlx6, mutations in other p63 target genes were also found to underlie several developmental disorders. For example, mutations in the p63 target gene P-cadherin cause ectodermal dysplasia, ectrodactyly, and macular dystrophy (EEM) and tricho-dento-osseous (TDO) syndrome (
      • Kjaer K.W.
      • Hansen L.
      • Schwabe G.C.
      • et al.
      Distinct CDH3 mutations cause ectodermal dysplasia, ectrodactyly, macular dystrophy (EEM syndrome).
      ;
      • Shimomura Y.
      • Wajid M.
      • Shapiro L.
      • et al.
      P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle.
      ). Limb abnormalities in patients with ectodermal dysplasia, ectrodactyly, and macular dystrophy show a remarkable similarity to those in patients with EEC (Figure 1). Further, ectodermal dysplasia, ectrodactyly, and macular dystrophy patients and EEC patients have hair abnormalities. As P-cadherin was previously found to be involved in hair follicle morphogenesis, these findings suggest that P-cadherin is an important mediator of p63 function during limb and hair development (
      • Jamora C.
      • DasGupta R.
      • Kocieniewski P.
      • et al.
      Links between signal transduction, transcription and adhesion in epithelial bud development.
      ). Another target gene of p63 that has been linked to a developmental disorder is Dlx3, mutations in which cause tricho-dento-osseous syndrome (
      • Price J.A.
      • Bowden D.W.
      • Wright J.T.
      • et al.
      Identification of a mutation in DLX3 associated with tricho-dento-osseous (TDO) syndrome.
      ;
      • Radoja N.
      • Guerrini L.
      • Lo Iacono N.
      • et al.
      Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias.
      ). In tricho-dento-osseous syndrome, the mutant Dlx3 proteins have a dominant-negative function toward the wild-type Dlx3 proteins, thus impairing the function of Dlx3 (
      • Duverger O.
      • Lee D.
      • Hassan M.Q.
      • et al.
      Molecular consequences of a frameshifted DLX3 mutant leading to tricho-dento-osseous syndrome.
      ). Interestingly, although p63 induces Dlx3 expression in basal keratinocytes, Dlx3 degrades p63 in suprabasal cell layers, suggesting that these proteins participate in an intricate feedback loop (
      • Di Costanzo A.
      • Festa L.
      • Duverger O.
      • et al.
      Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.
      ). Taken together, these data indicate that mutations in p63 itself and in genes transcriptionally regulated by p63 can lead to developmental disorders with similar phenotypes.

      Function of mutant p63 proteins

      All disorders caused by the p63 mutations identified to date are inherited in an autosomal dominant manner. However, it remains unclear how the mutant p63 proteins exert their dominant function. As the loss of one p63 allele does not cause ectodermal dysplasia in mice or humans, it appears that haplo-insufficiency is not the underlying disease mechanism (
      • Mills A.A.
      • Zheng B.
      • Wang X.J.
      • et al.
      p63 is a p53 homologue required for limb and epidermal morphogenesis.
      ;
      • Yang A.
      • Schweitzer R.
      • Sun D.
      • et al.
      p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.
      ;
      • van Bokhoven H.
      • Hamel B.C.
      • Bamshad M.
      • et al.
      p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation.
      ). Recent studies have provided some insight into the differential regulation of target genes by mutant p63 proteins expressed in patients. For example, whereas p63 proteins carrying AEC mutations can activate the Dlx5 and Dlx6 promoters, p63 proteins carrying EEC or SHFM mutations cannot, potentially providing an explanation for the lack of limb abnormalities in patients with AEC (
      • Lo Iacono N.
      • Mantero S.
      • Chiarelli A.
      • et al.
      Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects.
      ). However, these studies did not determine whether mutant p63 proteins affect the function of wild-type p63 proteins when they are co-expressed, as they are in ectodermal dysplasia patients. Thus, the function of mutant p63 proteins in the cellular context in which they are normally expressed is largely unknown. Further, the molecular basis for the differential target gene activation by different mutant p63 proteins remains unclear. However, it is likely that differences in DNA binding characteristics contribute to the differential activation of p63 target genes by different mutant p63 proteins. In fact, even though mutations in the p63 DNA binding domain occur only in SHFM and EEC, alterations in DNA binding characteristics are also predicted for some AEC mutants, because of conformational changes in the mutant p63 proteins (
      • McGrath J.A.
      • Duijf P.H.
      • Doetsch V.
      • et al.
      Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63.
      ). Another possible explanation for the differences in target gene activation may be the differential interaction of mutant p63 proteins with co-factors required for target gene activation or repression.

      Mouse models

      To further understand the role of p63 in developmental disorders, animal models that mimic these disorders are imperative. A mouse model that mimics the skin fragility phenotype observed in AEC patients was recently generated by downregulating ΔNp63 in the epidermis (
      • Koster M.I.
      • Dai D.
      • Marinari B.
      • et al.
      p63 induces key target genes required for epidermal morphogenesis.
      ). Both in mice with reduced epidermal ΔNp63 expression and in AEC patients, skin erosions were characterized by suprabasal epidermal proliferation, delayed terminal differentiation, and basement membrane abnormalities (
      • Koster M.I.
      • Marinari B.
      • Payne A.S.
      • et al.
      DeltaNp63 knockdown mice: a mouse model for AEC syndrome.
      ). Together, these abnormalities likely contribute to the skin fragility observed in AEC patients. Consistent with the finding that the skin fragility of AEC patients can be mimicked by downregulating ΔNp63, mutant ΔNp63α proteins expressed in AEC patients function as dominant-negative molecules, thereby preventing the induction of p63 target genes. Whether the additional developmental defects in AEC patients are also caused by a dominant-negative role of the mutant p63 proteins, or whether mutant p63 proteins also possess a gain-of-function role, remains to be determined. As the skin phenotypes of mice with reduced ΔNp63 expression and AEC patients are indistinguishable, this mouse model will be a valuable tool for testing therapeutic approaches that are aimed at treating skin erosions in AEC patients.

      p63 in Epidermal Development and Maintenance

      The human developmental disorders caused by alterations in the p63 pathway clearly demonstrate the importance of p63 in skin and appendage development. To dissect the genetic pathways controlled by p63, in vitro and in vivo models have been used (see below). These models have elucidated critical roles for p63 in the various key steps required for epidermal development and homeostasis (Figure 3).
      Figure thumbnail gr3
      Figure 3Role of p63 in the epidermis. The epidermis is a stratified epithelium, which consists of several layers of keratinocytes (indicated on the left). When interfollicular stem cells, which reside in the basal layer, divide, they give rise to transit amplifying (TA) cells that constitute most of the basal layer. When TA cells initiate terminal differentiation, they withdraw from the cell cycle and move suprabasally, forming the spinous layer. Further differentiation results in the formation of the granular and cornified cell layers. p63 is involved in the various processes required for epidermal development, differentiation, and homeostasis (indicated on the right). Genes involved in these processes are indicated in the text.

      Establishment of the epidermal fate

      p63 was initially identified on the basis of its sequence homology with the tumor suppressor gene p53 (
      • Yang A.
      • Kaghad M.
      • Wang Y.
      • et al.
      p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.
      ). However, unlike p53, p63 is expressed in a tissue-specific manner and is primarily expressed in stratified epithelia, such as the epidermis. Further, whereas p53-deficient mice are born without major abnormalities, ablating p63 from the germ line of mice led to striking developmental abnormalities. Mice lacking p63 were born with a shiny translucent skin and without any appendages, such as teeth, hair follicles, and mammary glands (
      • Mills A.A.
      • Zheng B.
      • Wang X.J.
      • et al.
      p63 is a p53 homologue required for limb and epidermal morphogenesis.
      ;
      • Yang A.
      • Schweitzer R.
      • Sun D.
      • et al.
      p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.
      ). The severe skin phenotype observed in p63-deficient mice was found to be caused by a complete failure of epidermal commitment, the process in which the surface ectoderm adopts an epidermal fate (
      • Koster M.I.
      • Kim S.
      • Mills A.A.
      • et al.
      p63 is the molecular switch for initiation of an epithelial stratification program.
      ;
      • De Rosa L.
      • Antonini D.
      • Ferone G.
      • et al.
      p63 suppresses non-epidermal lineage markers in a BMP dependent-manner via repression of SMAD7.
      ). During normal epidermal development, commitment to the epidermal lineage involves repression of the non-epidermal keratin pair K8 and K18 as well as induction of the epidermal keratin pair K5 and K14 (
      • Jackson B.W.
      • Grund C.
      • Winter S.
      • et al.
      Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.
      ;
      • Byrne C.
      • Tainsky M.
      • Fuchs E.
      Programming gene expression in developing epidermis.
      ). Interestingly, p63 is required both for the suppression of K8 and K18 and for the induction of K5 and K14. This was initially illustrated by the observation that K8 and K18 are aberrantly expressed in the surface epithelium of mice with a germ line deletion of p63, as well as in reconstructed human epidermis with decreased p63 expression levels (
      • Koster M.I.
      • Kim S.
      • Mills A.A.
      • et al.
      p63 is the molecular switch for initiation of an epithelial stratification program.
      ;
      • Truong A.B.
      • Kretz M.
      • Ridky T.W.
      • et al.
      p63 regulates proliferation and differentiation of developmentally mature keratinocytes.
      ;
      • De Rosa L.
      • Antonini D.
      • Ferone G.
      • et al.
      p63 suppresses non-epidermal lineage markers in a BMP dependent-manner via repression of SMAD7.
      ). Conversely, ectopic expression of p63 in cultured cells, single-layered epithelia, or embryonic stem cells led to the expression of K5 and K14 (
      • Koster M.I.
      • Kim S.
      • Mills A.A.
      • et al.
      p63 is the molecular switch for initiation of an epithelial stratification program.
      ;
      • Aberdam E.
      • Barak E.
      • Rouleau M.
      • et al.
      A pure population of ectodermal cells derived from human embryonic stem cells.
      ;
      • Medawar A.
      • Virolle T.
      • Rostagno P.
      • et al.
      DeltaNp63 is essential for epidermal commitment of embryonic stem cells.
      ;
      • Romano R.A.
      • Ortt K.
      • Birkaya B.
      • et al.
      An active role of the Delta N isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate.
      ). In addition to controlling the expression of keratins, p63 also directly represses two cell cycle inhibitors, Ink4a and Arf (
      • Su X.
      • Cho M.S.
      • Gi Y.J.
      • et al.
      Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.
      ). This repression is also critical for epidermal commitment, as shown by the finding that epidermal lineage commitment, indicated by the induction of K5 and K14 expression, in p63-deficient mice can be rescued by simultaneously ablating either Ink4a or Arf (
      • Su X.
      • Cho M.S.
      • Gi Y.J.
      • et al.
      Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.
      ). However, even though epidermal commitment is restored in these rescued mice, the epidermis does not mature normally, as shown by the heterogeneous expression of differentiation markers and the apparent absence of a stratum corneum (
      • Su X.
      • Cho M.S.
      • Gi Y.J.
      • et al.
      Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.
      ). Thus, epidermal differentiation requires the activation of additional genetic pathways that are not downstream of Ink4a and Arf.
      In addition to the failure to develop an epidermis, p63-deficient mice also fail to develop appendages. As appendage development requires extensive epithelial–mesenchymal interactions, the defects in appendage development in p63-deficient mice are likely to be, in part, secondary to the failure to develop an epidermis. However, p63 also has a direct role in appendage development by inducing genes that are required for the development of different appendages, such as Dlx5, Dlx6, and P-cadherin (
      • Lo Iacono N.
      • Mantero S.
      • Chiarelli A.
      • et al.
      Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects.
      ;
      • Shimomura Y.
      • Wajid M.
      • Shapiro L.
      • et al.
      P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle.
      ). This direct role of p63 in appendage development may provide an explanation for the existence of ectodermal dysplasias caused by p63 mutations in which appendages, but not the epidermis, are affected. In this subset of ectodermal dysplasias, mutant p63 proteins might still be able to activate p63 target genes required for normal development of the interfollicular epidermis, but they may fail to activate genes required for appendage development.

      p63 in basal keratinocytes

      In addition to its role in epidermal lineage commitment, p63 is also critical for epidermal differentiation and barrier formation, both during epidermal morphogenesis and postnatally (Figure 3). Within late embryonic and postnatal epidermis, the highest levels of p63 expression are observed in the basal layer, where it is predominantly expressed as the ΔNp63α isoform (
      • Yang A.
      • Kaghad M.
      • Wang Y.
      • et al.
      p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.
      ;
      • Liefer K.M.
      • Koster M.I.
      • Wang X.J.
      • et al.
      Down-regulation of p63 is required for epidermal UV-B-induced apoptosis.
      ). The basal layer is generally thought of as a homogeneous population of cells. However, it is actually composed of cells that are progressing along the differentiation pathway, including interfollicular stem cells, young TA cells, and mature TA cells. Whereas young TA cells are regularly cycling, mature TA cells have a limited proliferative capacity and are ready to embark on the terminal differentiation program (
      • Lehrer M.S.
      • Sun T.T.
      • Lavker R.M.
      Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.
      ). p63 may have very different functions in the various cell types within the basal layer. In fact, this would explain the apparent by contradictory results that have been obtained regarding the role of p63 in proliferation of basal keratinocytes. For example, p63 was found to maintain proliferation of basal keratinocytes, in part by repressing various cell cycle inhibitors, including p21, 14-3-3σ, Ink4a, and Arf, as well as by inducing the expression of genes required for cell cycle progression, including ADA and FASN (
      • Westfall M.D.
      • Mays D.J.
      • Sniezek J.C.
      • et al.
      The Delta Np63 alpha phosphoprotein binds the p21 and 14–3–3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations.
      ;
      • D'Erchia A.M.
      • Tullo A.
      • Lefkimmiatis K.
      • et al.
      The fatty acid synthase gene is a conserved p53 family target from worm to human.
      ;
      • Sbisa E.
      • Mastropasqua G.
      • Lefkimmiatis K.
      • et al.
      Connecting p63 to cellular proliferation: the example of the adenosine deaminase target gene.
      ;
      • Truong A.B.
      • Kretz M.
      • Ridky T.W.
      • et al.
      p63 regulates proliferation and differentiation of developmentally mature keratinocytes.
      ;
      • Lefkimmiatis K.
      • Caratozzolo M.F.
      • Merlo P.
      • et al.
      p73 and p63 Sustain cellular growth by transcriptional activation of cell cycle progression genes.
      ;
      • Su X.
      • Cho M.S.
      • Gi Y.J.
      • et al.
      Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.
      ). In apparent contrast, downregulating p63 in basal keratinocytes of mouse epidermis led to increased proliferation, showing that p63 is also required for cell cycle exit (
      • Koster M.I.
      • Dai D.
      • Marinari B.
      • et al.
      p63 induces key target genes required for epidermal morphogenesis.
      ). This induction of cell cycle exit is mediated, in part, by the direct induction of p57Kip2, a cyclin-dependent kinase inhibitor that is induced when keratinocytes undergo terminal differentiation (
      • Martinez L.A.
      • Chen Y.
      • Fischer S.M.
      • et al.
      Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation.
      ;
      • Beretta C.
      • Chiarelli A.
      • Testoni B.
      • et al.
      Regulation of the cyclin-dependent kinase inhibitor p57Kip2 expression by p63.
      ). Further, p63 directly represses the expression of genes required for cell cycle progression, including cyclin B2 and cdc2 (
      • Testoni B.
      • Mantovani R.
      Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63.
      ). This apparent controversy is most easily explained by postulating that p63 maintains proliferation in early TA cells, whereas it induces cell cycle exit in mature TA cells. This difference in p63 function may be caused by the different expression levels and/or interaction with co-factors that regulate p63 function.

      p63 in differentiation

      Whereas ΔNp63α expression is high in basal keratinocytes, its expression is reduced to approximately 25% in suprabasal keratinocytes (
      • King K.E.
      • Ponnamperuma R.M.
      • Gerdes M.J.
      • et al.
      Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis.
      ). The rapid downregulation of ΔNp63α in suprabasal keratinocytes seems to be mediated by several processes. First, ΔNp63α transcripts are degraded by microRNA-203 (
      • Lena A.M.
      • Shalom-Feuerstein R.
      • di Val Cervo P.R.
      • et al.
      miR-203 represses “/stemness/” by repressing [Delta] Np63.
      ;
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • et al.
      A skin microRNA promotes differentiation by repressing ‘stemness.
      ). As predicted, microRNA-203 is specifically expressed in suprabasal keratinocytes whereas it is virtually absent in basal keratinocytes (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of psoriasis?.
      ;
      • Lena A.M.
      • Shalom-Feuerstein R.
      • di Val Cervo P.R.
      • et al.
      miR-203 represses “/stemness/” by repressing [Delta] Np63.
      ;
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • et al.
      A skin microRNA promotes differentiation by repressing ‘stemness.
      ). Depletion of microRNA-203 was found to result in suprabasal proliferation of keratinocytes (
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • et al.
      A skin microRNA promotes differentiation by repressing ‘stemness.
      ). However, whether the increased proliferation was due to the observed increase in p63 expression or due to ectopic expression of other microRNA-203 target genes was not investigated. In addition to ΔNp63α transcript degradation, ΔNp63α protein is also actively degraded in suprabasal keratinocytes. This is mediated by several processes, including the targeting of ΔNp63α for degradation through the proteosomal pathways by the E3 ubiquitin ligase Itch and p14Arf (
      • Rossi M.
      • Aqeilan R.I.
      • Neale M.
      • et al.
      The E3 ubiquitin ligase Itch controls the protein stability of p63.
      ;
      • Vivo M.
      • Di C.A.
      • Fortugno P.
      • et al.
      Downregulation of DeltaNp63alpha in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation.
      ). Finally, the p63 target gene Dlx3 promotes ΔNp63α degradation in suprabasal keratinocytes (
      • Di Costanzo A.
      • Festa L.
      • Duverger O.
      • et al.
      Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.
      ).
      Together, the above-described mechanisms are responsible for the rapid functional inactivation of p63 in suprabasal keratinocytes. However, despite this active degradation of ΔNp63α in suprabasal cell layers, the remaining ΔNp63α protein is sufficient to control the important aspects of keratinocyte differentiation. For example, ΔNp63α directly induces IκB kinase α, a critical mediator of cell cycle exit during keratinocyte differentiation (
      • Koster M.I.
      • Dai D.
      • Marinari B.
      • et al.
      p63 induces key target genes required for epidermal morphogenesis.
      ;
      • Marinari B.
      • Ballaro C.
      • Koster M.I.
      • et al.
      IKK[alpha] is a p63 transcriptional target involved in the pathogenesis of ectodermal dysplasias.
      ). Further, ΔNp63α induces expression of the terminal differentiation marker K1 (
      • Nguyen B.C.
      • Lefort K.
      • Mandinova A.
      • et al.
      Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation.
      ;
      • Ogawa E.
      • Okuyama R.
      • Egawa T.
      • et al.
      p63/p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor.
      ). Finally, ΔNp63α is also important for the formation of the epidermal barrier by inducing at least two genes that are required for barrier formation, Claudin 1 and Alox12 (
      • Lopardo T.
      • Lo I.N.
      • Marinari B.
      • et al.
      Claudin-1 is a p63 target gene with a crucial role in epithelial development.
      ;
      • Kim S.
      • Choi I.F.
      • Quante J.R.
      • et al.
      p63 directly induces expression of Alox12, a regulator of epidermal barrier formation.
      ). Interestingly, despite the ability of ΔNp63α to induce genes required for terminal differentiation and barrier formation, it does not induce these genes in the basal layer. The most likely explanation for this observation is that co-factors required for the induction of terminal differentiation genes are absent in basal keratinocytes.

      p63 in cell–cell adhesion

      In addition to regulating epidermal development and differentiation, p63 is also important for cell–cell adhesion within the epidermis. A general role for p63 in mediating cell–cell adhesion was identified by downregulating p63 in cultured mammary cells (
      • Carroll D.K.
      • Carroll J.S.
      • Leong C.O.
      • et al.
      p63 regulates an adhesion programme and cell survival in epithelial cells.
      ). Downregulating p63 in this system led to impaired cell adhesion, as well as to reduced expression of several components of desmosomes, the multi-protein complexes that connect keratinocytes (
      • Cheng X.
      • Koch P.J.
      In vivo function of desmosomes.
      ;
      • Carroll D.K.
      • Carroll J.S.
      • Leong C.O.
      • et al.
      p63 regulates an adhesion programme and cell survival in epithelial cells.
      ). In addition, p63 was found to directly regulate the expression of the desmosomal component Perp (
      • Ihrie R.A.
      • Marques M.R.
      • Nguyen B.T.
      • et al.
      Perp is a p63-regulated gene essential for epithelial integrity.
      ). In the absence of Perp expression, mice display severe intra-epidermal blistering, further underscoring the importance of Perp in cell adhesion. Consistent with these findings, cell adhesion defects have also been reported in AEC patients, a subset of whom display aberrant Perp expression (
      • Payne A.S.
      • Yan A.C.
      • Ilyas E.
      • et al.
      Two novel TP63 mutations associated with the ankyloblepharon, ectodermal defects, and cleft lip and palate syndrome: a skin fragility phenotype.
      ;
      • Beaudry V.G.
      • Pathak N.
      • Koster M.I.
      • et al.
      Differential PERP regulation by TP63 mutants provides insight into AEC pathogenesis.
      ).

      p63 in basement membrane formation and cell-basement membrane adhesion

      In addition to its role in mediating cell–cell adhesion, p63 is also important for regulating the adhesion of keratinocytes to the basement membrane. Adhesion of keratinocytes to the basement membrane is mediated by integrins, a family of transmembrane receptors for the basement membrane protein laminin (
      • Larsen M.
      • Artym V.V.
      • Green J.A.
      • et al.
      The matrix reorganized: extracellular matrix remodeling and integrin signaling.
      ). p63 was found to induce the expression of several of these integrin subunits, including integrin α3 (
      • Kurata S.
      • Okuyama T.
      • Osada M.
      • et al.
      p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche.
      ;
      • Carroll D.K.
      • Carroll J.S.
      • Leong C.O.
      • et al.
      p63 regulates an adhesion programme and cell survival in epithelial cells.
      ). Further, p63 is also important for the formation of the basement membrane, as demonstrated by the basement membrane defects in mice with reduced ΔNp63 expression as well as in AEC patients (
      • Koster M.I.
      • Dai D.
      • Marinari B.
      • et al.
      p63 induces key target genes required for epidermal morphogenesis.
      ,
      • Koster M.I.
      • Marinari B.
      • Payne A.S.
      • et al.
      DeltaNp63 knockdown mice: a mouse model for AEC syndrome.
      ). p63 controls basement membrane formation, at least in part, by directly inducing the expression of the basement membrane component Fras1 (
      • Koster M.I.
      • Dai D.
      • Marinari B.
      • et al.
      p63 induces key target genes required for epidermal morphogenesis.
      ). Interestingly, in mice and humans, Fras1 deficiency causes a severe embryonic blistering phenotype, demonstrating the importance of Fras1 in basement membrane integrity (
      • McGregor L.
      • Makela V.
      • Darling S.M.
      • et al.
      Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.
      ;
      • Vrontou S.
      • Petrou P.
      • Meyer B.I.
      • et al.
      Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.
      ). Further, in prostate, the basement membrane associated with p63-expressing cells is thicker and more uniform than the basement membrane associated with basal cells that do not express p63 (
      • Liu A.
      • Wei L.
      • Gardner W.A.
      • et al.
      Correlated alterations in prostate basal cell layer and basement membrane.
      ). Together, these findings suggest a critical role for p63 in forming and maintaining the basement membrane.

      p63 in Stem Cells

      In addition to TA cells and differentiating keratinocytes, p63 is also expressed in the stem cells of several tissues, including the skin. However, a role for p63 in these stem cells has remained contentious. At least three different stem cell populations exist in the skin: interfollicular stem cells, bulge stem cells, and skin-derived precursors (SKPs). Of these, the interfollicular stem cells and the bulge stem cells regenerate the epidermis and hair follicle, respectively. Although either of these stem cell types can contribute to the epidermal or hair follicle lineage in response to injury, they only contribute to their own lineage under homeostatic conditions (
      • Claudinot S.
      • Nicolas M.
      • Oshima H.
      • et al.
      Long-term renewal of hair follicles from clonogenic multipotent stem cells.
      ;
      • Ito M.
      • Liu Y.
      • Yang Z.
      • et al.
      Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.
      ). A functional role for p63 in interfollicular and bulge stem cells was first proposed on the basis of the phenotype of p63-deficient mice. Some investigators attributed the absence of epidermal morphogenesis in these mice to a premature depletion of stem cells (
      • Yang A.
      • Schweitzer R.
      • Sun D.
      • et al.
      p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.
      ). Further, colony-forming assays using keratinocytes with reduced p63 expression levels resulted in the formation of smaller colonies, which has been interpreted as evidence for stem cell depletion (
      • Senoo M.
      • Pinto F.
      • Crum C.P.
      • et al.
      p63 Is essential for the proliferative potential of stem cells in stratified epithelia.
      ). However, whether this apparent lack of proliferation affected stem cells and/or TA cells was not investigated.
      Recently, it was shown that TAp63 isoforms are also expressed in SKPs, dermal precursor cells that reside in the dermal sheath and dermal papilla (
      • Fernandes K.J.L.
      • McKenzie I.A.
      • Mill P.
      • et al.
      A dermal niche for multipotent adult skin-derived precursor cells.
      ;
      • Su X.
      • Paris M.
      • Gi Y.J.
      • et al.
      TAp63 prevents premature aging by promoting adult stem cell maintenance.
      ). Interestingly, using a conditional TAp63 knockout mouse model, it was found that ablation of TAp63 from both the dermal and epidermal compartments led to skin blistering and accelerated aging (
      • Su X.
      • Paris M.
      • Gi Y.J.
      • et al.
      TAp63 prevents premature aging by promoting adult stem cell maintenance.
      ). However, ablation of TAp63 specifically in the epidermis did not recapitulate these phenotypes, suggesting that TAp63 exerts its role in SKPs and not in the epidermis (
      • Su X.
      • Paris M.
      • Gi Y.J.
      • et al.
      TAp63 prevents premature aging by promoting adult stem cell maintenance.
      ). However, as ablation of TAp63 specifically from SKPs is currently not technically feasible, the ultimate proof for a role of TAp63 in SKPs has yet to be generated.

      Summary

      The involvement of abnormalities in the p63 pathway in a subset of developmental disorders underscores the importance of p63 in normal skin and appendage development. Indeed, animal models and cell culture studies have identified the roles of p63 in various processes, including epidermal lineage commitment, epidermal differentiation, cell adhesion, and basement membrane formation. However, the function of mutant p63 proteins expressed in ectodermal dysplasia remains poorly understood. Whereas mutant p63 proteins in some ectodermal dysplasias such as AEC cause skin fragility, mutant p63 proteins expressed in other ectodermal dysplasias such as EEC cause limb abnormalities. These differences are likely caused by differential activation of p63 target genes by the different mutant p63 proteins. For example, whereas EEC mutants may be able to normally activate p63 target genes involved in skin development and differentiation, they may fail to activate target genes required for limb development. Further studies into the role of p63 in skin and appendage development, as well as into the molecular role of mutant p63 proteins, will be necessary for a better understanding of the role of p63 in ectodermal dysplasias and other developmental disorders.

      ACKNOWLEDGMENTS

      I thank Dr Peter J Koch for his constructive comments on this paper and the National Foundation for Ectodermal Dysplasias (NFED) for the images shown in Figure 1. Work in my laboratory is supported by the National Institutes of Health (AR054696), the American Skin Association (ASA), and the NFED.

      REFERENCES

        • Aberdam E.
        • Barak E.
        • Rouleau M.
        • et al.
        A pure population of ectodermal cells derived from human embryonic stem cells.
        Stem Cells. 2008; 26: 440-444
        • Beaudry V.G.
        • Pathak N.
        • Koster M.I.
        • et al.
        Differential PERP regulation by TP63 mutants provides insight into AEC pathogenesis.
        Am J Med Genet A. 2009; 149A: 1952-1957
        • Beretta C.
        • Chiarelli A.
        • Testoni B.
        • et al.
        Regulation of the cyclin-dependent kinase inhibitor p57Kip2 expression by p63.
        Cell Cycle. 2005; 4: 1625-1631
        • Bree A.F.
        Clinical lessons learned from the International Research Symposium on Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome.
        Am J Med Genet A. 2009; 149A: 1894-1899
        • Brunner H.G.
        • Hamel B.C.J.
        • van Bokhoven H.
        The p63 gene in EEC and other syndromes.
        J Med Genet. 2002; 39: 377-381
        • Byrne C.
        • Tainsky M.
        • Fuchs E.
        Programming gene expression in developing epidermis.
        Development. 1994; 120: 2369-2383
        • Carroll D.K.
        • Carroll J.S.
        • Leong C.O.
        • et al.
        p63 regulates an adhesion programme and cell survival in epithelial cells.
        Nat Cell Biol. 2006; 8: 551-561
        • Celli J.
        • Duijf P.
        • Hamel B.C.
        • et al.
        Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.
        Cell. 1999; 99: 143-153
        • Cheng X.
        • Koch P.J.
        In vivo function of desmosomes.
        J Dermatol. 2004; 31: 171-187
        • Claudinot S.
        • Nicolas M.
        • Oshima H.
        • et al.
        Long-term renewal of hair follicles from clonogenic multipotent stem cells.
        Proc Natl Acad Sci USA. 2005; 102: 14677-14682
        • D'Erchia A.M.
        • Tullo A.
        • Lefkimmiatis K.
        • et al.
        The fatty acid synthase gene is a conserved p53 family target from worm to human.
        Cell Cycle. 2006; 5: 750-758
        • De Rosa L.
        • Antonini D.
        • Ferone G.
        • et al.
        p63 suppresses non-epidermal lineage markers in a BMP dependent-manner via repression of SMAD7.
        J Biol Chem. 2009; 284: 30574-30582
        • Di Costanzo A.
        • Festa L.
        • Duverger O.
        • et al.
        Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.
        Cell Cycle. 2009; 8: 1185-1195
        • Duijf P.H.G.
        • van Bokhoven H.
        • Brunner H.G.
        Pathogenesis of split-hand/split-foot malformation.
        Hum Mol Genet. 2003; 12: R51-R60
        • Duverger O.
        • Lee D.
        • Hassan M.Q.
        • et al.
        Molecular consequences of a frameshifted DLX3 mutant leading to tricho-dento-osseous syndrome.
        J Biol Chem. 2008; 283: 20198-20208
        • Fernandes K.J.L.
        • McKenzie I.A.
        • Mill P.
        • et al.
        A dermal niche for multipotent adult skin-derived precursor cells.
        Nat Cell Biol. 2004; 6: 1082-1093
        • Fuchs E.
        Finding one's niche in the skin.
        Cell Stem Cell. 2009; 4: 499-502
        • Ianakiev P.
        • Kilpatrick M.W.
        • Toudjarska I.
        • et al.
        Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27.
        Am J Hum Genet. 2000; 67: 59-66
        • Ihrie R.A.
        • Marques M.R.
        • Nguyen B.T.
        • et al.
        Perp is a p63-regulated gene essential for epithelial integrity.
        Cell. 2005; 120: 843-856
        • Ito M.
        • Liu Y.
        • Yang Z.
        • et al.
        Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.
        Nat Med. 2005; 11: 1351-1354
        • Jackson B.W.
        • Grund C.
        • Winter S.
        • et al.
        Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.
        Differentiation. 1981; 20: 203-216
        • Jamora C.
        • DasGupta R.
        • Kocieniewski P.
        • et al.
        Links between signal transduction, transcription and adhesion in epithelial bud development.
        Nature. 2003; 422: 317-322
        • Julapalli M.R.
        • Scher R.K.
        • Sybert V.P.
        • et al.
        Dermatologic findings of ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome.
        Am J Med Genet A. 2009; 149A: 1900-1906
        • Kim S.
        • Choi I.F.
        • Quante J.R.
        • et al.
        p63 directly induces expression of Alox12, a regulator of epidermal barrier formation.
        Exp Dermatol. 2009; 18: 1016-1021
        • King K.E.
        • Ponnamperuma R.M.
        • Gerdes M.J.
        • et al.
        Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis.
        Carcinogenesis. 2006; 27: 53-63
        • Kjaer K.W.
        • Hansen L.
        • Schwabe G.C.
        • et al.
        Distinct CDH3 mutations cause ectodermal dysplasia, ectrodactyly, macular dystrophy (EEM syndrome).
        J Med Genet. 2005; 42: 292-298
        • Koster M.I.
        • Dai D.
        • Marinari B.
        • et al.
        p63 induces key target genes required for epidermal morphogenesis.
        Proc Natl Acad Sci USA. 2007; 104: 3255-3260
        • Koster M.I.
        • Kim S.
        • Mills A.A.
        • et al.
        p63 is the molecular switch for initiation of an epithelial stratification program.
        Genes Dev. 2004; 18: 126-131
        • Koster M.I.
        • Marinari B.
        • Payne A.S.
        • et al.
        DeltaNp63 knockdown mice: a mouse model for AEC syndrome.
        Am J Med Genet A. 2009; 149A: 1942-1947
        • Koster M.I.
        • Roop D.R.
        Mechanisms regulating epithelial stratification.
        Annu Rev Cell Dev Biol. 2007; 23: 93-113
        • Kurata S.
        • Okuyama T.
        • Osada M.
        • et al.
        p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche.
        J Biol Chem. 2004; 279: 50069-50077
        • Larsen M.
        • Artym V.V.
        • Green J.A.
        • et al.
        The matrix reorganized: extracellular matrix remodeling and integrin signaling.
        Curr Opin Cell Biol. 2006; 18: 463-471
        • Lefkimmiatis K.
        • Caratozzolo M.F.
        • Merlo P.
        • et al.
        p73 and p63 Sustain cellular growth by transcriptional activation of cell cycle progression genes.
        Cancer Res. 2009; 69: 8563-8571
        • Lehrer M.S.
        • Sun T.T.
        • Lavker R.M.
        Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.
        J Cell Sci. 1998; 111: 2867-2875
        • Lena A.M.
        • Shalom-Feuerstein R.
        • di Val Cervo P.R.
        • et al.
        miR-203 represses “/stemness/” by repressing [Delta] Np63.
        Cell Death Differ. 2008; 15: 1187-1195
        • Liefer K.M.
        • Koster M.I.
        • Wang X.J.
        • et al.
        Down-regulation of p63 is required for epidermal UV-B-induced apoptosis.
        Cancer Res. 2000; 60: 4016-4020
        • Liu A.
        • Wei L.
        • Gardner W.A.
        • et al.
        Correlated alterations in prostate basal cell layer and basement membrane.
        Int J Biol Sci. 2009; 5: 276-285
        • Lo Iacono N.
        • Mantero S.
        • Chiarelli A.
        • et al.
        Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects.
        Development. 2008; 135: 1377-1388
        • Lopardo T.
        • Lo I.N.
        • Marinari B.
        • et al.
        Claudin-1 is a p63 target gene with a crucial role in epithelial development.
        PLoS ONE. 2008; 3: e2715
        • Mangiulli M.
        • Valletti A.
        • Caratozzolo M.F.
        • et al.
        Identification and functional characterization of two new transcriptional variants of the human p63 gene.
        Nucl Acids Res. 2009; 37: 6092-6104
        • Marinari B.
        • Ballaro C.
        • Koster M.I.
        • et al.
        IKK[alpha] is a p63 transcriptional target involved in the pathogenesis of ectodermal dysplasias.
        J Invest Dermatol. 2008; 129: 60-69
        • Martinez L.A.
        • Chen Y.
        • Fischer S.M.
        • et al.
        Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation.
        Oncogene. 1999; 18: 397-406
        • McGrath J.A.
        • Duijf P.H.
        • Doetsch V.
        • et al.
        Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63.
        Hum Mol Genet. 2001; 10: 221-229
        • McGregor L.
        • Makela V.
        • Darling S.M.
        • et al.
        Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.
        Nat Genet. 2003; 34: 203-208
        • Medawar A.
        • Virolle T.
        • Rostagno P.
        • et al.
        DeltaNp63 is essential for epidermal commitment of embryonic stem cells.
        PLoS ONE. 2008; 3: e3441
        • Merlo G.R.
        • Paleari L.
        • Mantero S.
        • et al.
        Mouse model of split hand/foot malformation type I.
        Genesis. 2002; 33: 97-101
        • Mills A.A.
        • Zheng B.
        • Wang X.J.
        • et al.
        p63 is a p53 homologue required for limb and epidermal morphogenesis.
        Nature. 1999; 398: 708-713
        • Nguyen B.C.
        • Lefort K.
        • Mandinova A.
        • et al.
        Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation.
        Genes Dev. 2006; 20: 1028-1042
        • Ogawa E.
        • Okuyama R.
        • Egawa T.
        • et al.
        p63/p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor.
        J Biol Chem. 2008; 283: 34241-34249
        • Payne A.S.
        • Yan A.C.
        • Ilyas E.
        • et al.
        Two novel TP63 mutations associated with the ankyloblepharon, ectodermal defects, and cleft lip and palate syndrome: a skin fragility phenotype.
        Arch Dermatol. 2005; 141: 1567-1573
        • Price J.A.
        • Bowden D.W.
        • Wright J.T.
        • et al.
        Identification of a mutation in DLX3 associated with tricho-dento-osseous (TDO) syndrome.
        Hum Mol Genet. 1998; 7: 563-569
        • Radoja N.
        • Guerrini L.
        • Lo Iacono N.
        • et al.
        Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias.
        Development. 2007; 134: 13-18
        • Rice R.H.
        • Green H.
        The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein.
        Cell. 1977; 11: 417-422
        • Rinne T.
        • Brunner H.G.
        • van Bokhoven H.
        p63-associated disorders.
        Cell Cycle. 2007; 6: 262-268
        • Rinne T.
        • Clements S.E.
        • Lamme E.
        • et al.
        A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp-Hodgkin/Hay-Wells-like syndromes.
        Hum Mol Genet. 2008; 17: 1968-1977
        • Robledo R.F.
        • Rajan L.
        • Li X.
        • et al.
        The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development.
        Genes Dev. 2002; 16: 1089-1101
        • Romano R.A.
        • Ortt K.
        • Birkaya B.
        • et al.
        An active role of the Delta N isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate.
        PLoS ONE. 2009; 4: e5623
        • Rossi M.
        • Aqeilan R.I.
        • Neale M.
        • et al.
        The E3 ubiquitin ligase Itch controls the protein stability of p63.
        Proc Natl Acad Sci USA. 2006; 103: 12753-12758
        • Sbisa E.
        • Mastropasqua G.
        • Lefkimmiatis K.
        • et al.
        Connecting p63 to cellular proliferation: the example of the adenosine deaminase target gene.
        Cell Cycle. 2006; 5: 205-212
        • Scherer S.
        • Poorka] P.
        • Massa H.
        • et al.
        Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly.
        Hum Mol Genet. 1994; 3: 1345-1354
        • Senoo M.
        • Pinto F.
        • Crum C.P.
        • et al.
        p63 Is essential for the proliferative potential of stem cells in stratified epithelia.
        Cell. 2007; 129: 523-536
        • Shimomura Y.
        • Wajid M.
        • Shapiro L.
        • et al.
        P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle.
        Development. 2008; 135: 743-753
        • Sonkoly E.
        • Wei T.
        • Janson P.C.
        • et al.
        MicroRNAs: novel regulators involved in the pathogenesis of psoriasis?.
        PLoS ONE. 2007; 2: e610
        • Steven A.C.
        • Steinert P.M.
        Protein composition of cornified cell envelopes of epidermal keratinocytes.
        J Cell Sci. 1994; 107: 693-700
        • Su X.
        • Paris M.
        • Gi Y.J.
        • et al.
        TAp63 prevents premature aging by promoting adult stem cell maintenance.
        Cell Stem Cell. 2009; 5: 64-75
        • Su X.
        • Cho M.S.
        • Gi Y.J.
        • et al.
        Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf.
        EMBO J. 2009; 28: 1904-1915
        • Sutton V.R.
        • Plunkett K.
        • Dang D.X.
        • et al.
        Craniofacial and anthropometric phenotype in ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (Hay-Wells syndrome) in a cohort of 17 patients.
        Am J Med Genet A. 2009; 149A: 1916-1921
        • Testoni B.
        • Mantovani R.
        Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63.
        Nucl Acids Res. 2006; 34: 928-938
        • Truong A.B.
        • Kretz M.
        • Ridky T.W.
        • et al.
        p63 regulates proliferation and differentiation of developmentally mature keratinocytes.
        Genes Dev. 2006; 20: 3185-3197
        • van Bokhoven H.
        • Hamel B.C.
        • Bamshad M.
        • et al.
        p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation.
        Am J Hum Genet. 2001; 69: 481-492
        • Vivo M.
        • Di C.A.
        • Fortugno P.
        • et al.
        Downregulation of DeltaNp63alpha in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation.
        Cell Cycle. 2009; 8: 3537-3543
        • Vrontou S.
        • Petrou P.
        • Meyer B.I.
        • et al.
        Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.
        Nat Genet. 2003; 34: 209-214
        • Westfall M.D.
        • Mays D.J.
        • Sniezek J.C.
        • et al.
        The Delta Np63 alpha phosphoprotein binds the p21 and 14–3–3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations.
        Mol Cell Biol. 2003; 23: 2264-2276
        • Yang A.
        • Kaghad M.
        • Wang Y.
        • et al.
        p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.
        Mol Cell. 1998; 2: 305-316
        • Yang A.
        • Schweitzer R.
        • Sun D.
        • et al.
        p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.
        Nature. 1999; 398: 714-718
        • Yi R.
        • Poy M.N.
        • Stoffel M.
        • et al.
        A skin microRNA promotes differentiation by repressing ‘stemness.
        Nature. 2008; 452: 225-229