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• W1812133033 abstract "Research Article13 January 2012Open Access Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome Giustina Ferone Giustina Ferone Fondazione IRCCS SDN, Napoli, Italy Search for more papers by this author Helen A. Thomason Helen A. Thomason Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Current address: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom Search for more papers by this author Dario Antonini Dario Antonini CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Laura De Rosa Laura De Rosa CEINGE Biotecnologie Avanzate, Napoli, Italy Current address: Center for Regenerative Medicine Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Bing Hu Bing Hu Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Marica Gemei Marica Gemei CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Huiqing Zhou Huiqing Zhou Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Raffaele Ambrosio Raffaele Ambrosio Fondazione IRCCS SDN, Napoli, Italy Search for more papers by this author David P. Rice David P. Rice Institute of Dentistry, University of Helsinki and Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Dario Acampora Dario Acampora CEINGE Biotecnologie Avanzate, Napoli, Italy Institute of Genetics and Biophysics CNR, Napoli, Italy Search for more papers by this author Hans van Bokhoven Hans van Bokhoven Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Luigi Del Vecchio Luigi Del Vecchio CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Maranke I. Koster Maranke I. Koster Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Search for more papers by this author Gianluca Tadini Gianluca Tadini Center of Inherited Cutaneous Diseases, University of Milan, Fondazione Osp Maggiore Policlinico Ca' Granda, Milano, Italy Search for more papers by this author Bradley Spencer-Dene Bradley Spencer-Dene Experimental Pathology Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Michael Dixon Michael Dixon Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Search for more papers by this author Jill Dixon Jill Dixon Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Search for more papers by this author Caterina Missero Corresponding Author Caterina Missero [email protected] CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Giustina Ferone Giustina Ferone Fondazione IRCCS SDN, Napoli, Italy Search for more papers by this author Helen A. Thomason Helen A. Thomason Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Current address: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom Search for more papers by this author Dario Antonini Dario Antonini CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Laura De Rosa Laura De Rosa CEINGE Biotecnologie Avanzate, Napoli, Italy Current address: Center for Regenerative Medicine Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Bing Hu Bing Hu Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Marica Gemei Marica Gemei CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Huiqing Zhou Huiqing Zhou Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Raffaele Ambrosio Raffaele Ambrosio Fondazione IRCCS SDN, Napoli, Italy Search for more papers by this author David P. Rice David P. Rice Institute of Dentistry, University of Helsinki and Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Dario Acampora Dario Acampora CEINGE Biotecnologie Avanzate, Napoli, Italy Institute of Genetics and Biophysics CNR, Napoli, Italy Search for more papers by this author Hans van Bokhoven Hans van Bokhoven Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Luigi Del Vecchio Luigi Del Vecchio CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Maranke I. Koster Maranke I. Koster Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Search for more papers by this author Gianluca Tadini Gianluca Tadini Center of Inherited Cutaneous Diseases, University of Milan, Fondazione Osp Maggiore Policlinico Ca' Granda, Milano, Italy Search for more papers by this author Bradley Spencer-Dene Bradley Spencer-Dene Experimental Pathology Laboratory, Cancer Research UK London Research Institute, London, UK Search for more papers by this author Michael Dixon Michael Dixon Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Search for more papers by this author Jill Dixon Jill Dixon Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom Search for more papers by this author Caterina Missero Corresponding Author Caterina Missero [email protected] CEINGE Biotecnologie Avanzate, Napoli, Italy Search for more papers by this author Author Information Giustina Ferone1, Helen A. Thomason2,11, Dario Antonini3, Laura De Rosa3,12, Bing Hu4, Marica Gemei3, Huiqing Zhou5, Raffaele Ambrosio1, David P. Rice6, Dario Acampora3,7, Hans van Bokhoven5, Luigi Del Vecchio3, Maranke I. Koster8, Gianluca Tadini9, Bradley Spencer-Dene10, Michael Dixon2, Jill Dixon2 and Caterina Missero *,3 1Fondazione IRCCS SDN, Napoli, Italy 2Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom 3CEINGE Biotecnologie Avanzate, Napoli, Italy 4Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 5Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 6Institute of Dentistry, University of Helsinki and Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland 7Institute of Genetics and Biophysics CNR, Napoli, Italy 8Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 9Center of Inherited Cutaneous Diseases, University of Milan, Fondazione Osp Maggiore Policlinico Ca' Granda, Milano, Italy 10Experimental Pathology Laboratory, Cancer Research UK London Research Institute, London, UK 11Current address: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom 12Current address: Center for Regenerative Medicine Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy *Tel: +39 081 3737853; Fax: +39 081 3737808 EMBO Mol Med (2012)4:192-205https://doi.org/10.1002/emmm.201100199 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63+/L514F) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b−/− mice. Restoring Fgfr2b expression in p63+/L514F epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome. See accompanying article http://dx.doi.org/10.1002/emmm.201100202 The paper explained PROBLEM: Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome is an autosomal dominant disorder mainly characterized by cleft palate, and ectodermal dysplasia associated with severe involvement of the skin at birth, whose consequences are life-threatening. It is caused by heterozygous mutations in the p63 gene, encoding a transcription factor essential for the development of several stratified epithelia. Full understanding of AEC syndrome is impaired by genetic and clinical variability among unrelated patients, and by lack of foetal or neonatal biological samples. RESULTS: Our article reports the characterization of a knock-in mouse model for AEC syndrome that recapitulates the human disorder both at the genetic and phenotypic levels. The p63 AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting the expression of a number of p63 target genes, including Fgfr2 and Fgfr3. Impairment of FGF signalling in p63 mutant embryos causes epidermal hypoplasia and cleft palate due to reduced expansion of ectodermal progenitor cells. These phenotypes are associated with a defective stem cell compartment that is observed not only in AEC mutant mice and in mice lacking epithelial Fgfr2, but also in humans affected by AEC syndrome. IMPACT: Our studies provide novel mechanistic insights into the pathogenesis of AEC syndrome and establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells during embryonic development, a step required for skin and palate morphogenesis. Moreover, our observations provide the first evidence that reduced FGF signalling during embryogenesis leads to defects in the epidermal stem cell compartment. INTRODUCTION The p63 gene encodes a tetrameric transcription factor belonging to the p53 family, which has an essential function in the formation of stratified epithelia. p63 expression is driven by two independent promoters generating TA and ΔN classes of proteins, each producing α, β or γ ends as the result of alternative splicing events towards the C-terminal region. The C-terminal end of the α isoform contains a sterile-alpha-motif (SAM) domain and a transactivation inhibitory domain, which are present in p63 and in p73 but absent from p53 (Yang et al, 1998). p63 is expressed most abundantly in the basal regenerative layers of stratified epithelia, where ΔNp63α, that can function either as an activator or a repressor, is the predominant isoform (Koster et al, 2007; Leboeuf et al, 2011). Mice lacking the p63 gene die soon after birth with severe defects of all stratified epithelia and their derivatives, facial clefting and impaired limb formation (Mills et al, 1999; Yang et al, 1999). Genome-wide profiling of p63 binding regions and gene expression analyses have revealed that p63 directly regulates a large number of genes (Della Gatta et al, 2008; Kouwenhoven et al, 2010; Vigano et al, 2006; Yang et al, 2006). p63 is critical for a number of cellular and developmental processes in stratified epithelia, which include promoting cell proliferation (Antonini et al, 2010; Senoo et al, 2007; Truong et al, 2006), cell adhesion (Carroll et al, 2006; Koster et al, 2007) and stratification (Koster et al, 2004; Truong et al, 2006), while at the same time suppressing terminal differentiation (Nguyen et al, 2006). In addition, p63 is required, at least in vitro, for the proliferative potential of self-renewing populations of corneal and epidermal stem cells (Rama et al, 2010; Senoo et al, 2007) and in vivo for thymic epithelial cells (Senoo et al, 2007). At least five human malformation syndromes resulting from heterozygous mutations in p63 exhibit phenotypes that are reminiscent of those displayed by p63−/− mice although they are less severe. Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome (or Hay–Wells syndrome; OMIM 106260) is caused by mutations clustered mostly in the SAM domain. AEC syndrome differs from other conditions resulting from p63 mutations, in the severity of the skin phenotype, the occurrence of ankyloblepharon and the absence of ectrodactyly (Dishop et al, 2009; McGrath et al, 2001). Dermatological features include mild atrophy, often associated with congenital erythroderma, widespread skin erosions at or soon after birth and ectodermal dysplasia (Dishop et al, 2009; Fete et al, 2009; Julapalli et al, 2009). Investigation of the pathogenesis of AEC syndrome has been hampered by the lack of an animal model closely resembling the human disorder. To this aim, we generated the p63+/L514F mouse, a faithful mouse model of AEC syndrome, which is characterized by hypoplastic and fragile skin, ectodermal dysplasia and cleft palate. We find that epidermal hypoplasia and cleft palate are associated with a transient reduction in epithelial cell proliferation during development. These defects closely resemble those observed in the Fgfr2b−/− mice (De Moerlooze et al, 2000; Petiot et al, 2003; Rice et al, 2004). p63 transcriptionally controls the FGF receptors Fgfr2 and Fgfr3 and their expression as well as downstream signalling is affected in p63+/L514F mutant mice. We propose that impaired FGF signalling downstream of p63 is likely an important determinant of reduced ectodermal cell proliferation and defective self-renewing compartment in AEC syndrome. RESULTS The phenotype of p63+/L514F mice mimics that of AEC syndrome To characterize the developmental alterations that occur in AEC syndrome, we generated a knock-in mouse model carrying a leucine to phenylalanine substitution in position 514 (L514F) in the p63 protein (Fig 1A–D). L514 is a highly conserved amino acid in the first helix of the SAM domain, which is mutated to either phenylalanine or valine in AEC patients (McGrath et al, 2001; Payne et al, 2005; Supporting Information Fig S1A). A correctly targeted embryonic stem cell line allowed the mutation to be transmitted through germline to produce heterozygous p63+/L514F mice. p63 messenger RNA (mRNA) was expressed at similar levels in p63+/L514F mutant and in wild-type epidermis (Supporting Information Fig S1B), whereas p63 protein was more abundant in mutant than in wild-type epidermis (Supporting Information Fig S1C-D) consistent with the previously reported p63 accumulation in the skin of AEC patients (Browne et al, 2011; Moretti et al, 2010). No aberrant isoforms were detected either at the RNA or the protein levels (Supporting Information Fig S1C and S1E). Figure 1. Generation and phenotype of p63+/L514F mice. A.. Gene targeting strategy used to generate the p63+/L514F knock-in mice. The L514F mutation is indicated with *. Dotted black lines indicate the sizes of the fragments generated from each allele upon EcoRI (E) digestion. Oligonucleotide primers (arrows) used for PCR analysis and the relative PCR product length, are indicated. B.. Southern blot analysis of ES cell clones using the EcoRI digestion of genomic DNA and the 5′ probe upstream of the recombinant targeted site. C.. DNA sequencing analysis of the L514F point mutation in properly targeted ES cells. D.. In vivo removal of the NEO cassette obtained by crossing chimeras with CMV-Cre transgenic mice, as revealed by PCR analysis performed on tail genomic DNA using one forward (F) and two reverse primers (R1 and R2). E.. H&E staining of sagittal sections of +/+ and p63+/L514F newborn heads. Cleft of the secondary palate is indicated by the arrow. F.. H&E staining of dorsal skin of p63+/L514F mice at P0. Mutant epidermis is thinner and with less developed hair follicles. G.. Three-dimensional computer-aided reconstruction of tooth crown morphology of the first lower molar at P0. The cusps are numbered as buccal (B1, 2, 3), lingual (L1, 2, 3) and distal (4). The axis of the tooth coming in contact with adjacent teeth is indicated: mesial (M) and distal (D). Scale bar: 400 µm. H.. Histological analysis of sagittal sections of the first lower molar at P0. EO, enamel organ; DP, dental papilla; AM, ameloblasts; OD, odontoblasts. Scale bar: upper panel 50 µm, lower panel 40 µm. Download figure Download PowerPoint Visual inspection at birth revealed that newborn p63+/L514F mice exhibited air accumulation in their stomach and intestine without milk uptake and the mice died shortly after birth. p63+/L514F mice (n = 187) exhibited a fully penetrant wide cleft of the secondary palate without involvement of the lip/primary palate or other cranial or mandibular malformations (Fig 1E; Supporting Information Fig S2A–C). Cleft palate was not rescued by crossing chimeras with females of different inbred strains (see Supporting information) suggesting that this defect was independent of the genetic background. Ankyloblepharon-ectodermal defects-cleft lip/palate patients are characterized by mild atrophy of the skin, skin erosions and sparse or absent hair (Dishop et al, 2009; McGrath et al, 2001). p63+/L514F mice displayed reduced skin folding and a translucent appearance of the skin at birth (Supporting Information Fig S3A). At the histological level, mutant skin was thinner than that of wild-type mice with consistently thin epidermis and dermis (Fig 1F). Blistering skin lesions, localized mainly between the basal and suprabasal layers of the epidermis, were observed in a fraction of mutant mice (21%; n = 28) (Supporting Information Fig S3B). The number of hair follicles in more advanced stages of differentiation was reduced (Fig 1F and Supporting Information Fig S3C). In spite of normal hair placode formation, hair follicle morphogenesis was delayed (Supporting Information Fig S3C-D). Vibrissae were similarly shortened (Supporting Information Fig S3E). Among the dental abnormalities observed in AEC patients are smaller teeth, with small occlusal surface in premolars and molars (Farrington & Lausten, 2009). Similarly, in p63+/L514F mice the crown of the lower first molar tooth was smaller than the wild-type, particularly in the mesial–distal axis, and the distal cusp was missing (Fig 1G). In addition, the tooth germ was smaller with less developed cusps and with hypoplastic pre-ameloblast and odontoblast layers (Fig 1H). Ankyloblepharon, a characteristic defect in AEC patients, could not be evaluated in mutant mice as eyelids are normally fused until postnatal day 15 (P15). Taken together, these data show that p63+/L514F heterozygous mice phenocopy many aspects of the human AEC syndrome and thus represent the first mouse model for this disease. Palate hypoplasia and reduced cell proliferation of the palatal epithelium in p63+/L514F mice To identify the cause of cleft palate, we analyzed the p63+/L514F mouse oral cavity during development. At embryonic day (E)13.5, although reproducibly smaller, the mutant palatal shelves pointed downwards on either side of the tongue similar to their wild-type littermates (Fig 2A, C and D). Although the mutant palatal shelves elevated appropriately at E14.5, they were smaller, widely spaced and failed to meet in the midline (Fig 2B). Figure 2. Palatal shelf hypoplasia and reduced epithelial cell proliferation in p63+/L514F embryos. A.. H&E staining of coronal sections of p63+/L514F mid palate at E13.5 reveals grossly similar morphology of palatal shelves compared to controls. t, tongue; p, palatal shelf; tb, molar tooth bud; m, mandible. Scale bar: 200 µm. B.. H&E and Alcian Blue staining of coronal sections of p63+/L514F mid palate at E14.5 reveals a failure of the palatal shelves to meet in the horizontal plane. nc, nasal cavity. Scale bar: 200 µm. C.. A closer view of H&E staining of E13.5 coronal sections reveals that mutant palatal shelves are hypoplastic compared to controls. Scale bar: 50 µm. D.. Quantification of the anterior, mid and posterior palatal shelf area. Error bars represent standard deviation (SD). Anterior palate *p-value = 0.00032; n = 9. Mid palate *p-value = 0.00079; n = 9. Posterior palate p-value = 0.0097; n = 9. E.. Immunofluorescence with anti-BrdU antibodies at E13 (left panel) reveals reduced proliferation of mutant palatal shelves. F.. The average percentage of BrdU incorporation is reduced in mutant palatal epithelium at E13 and E13.5. Epithelium at E13 p-value = 0.009; n = 9. Mesenchyme at E13 p-value = 0.032; n = 9. Epithelium at E13.5 p-value = 0.018; n = 9. Data are represented as mean ± SD. Scale bar: 50 µm. G.. Double immunofluorescence staining for the indicated markers reveals hypoplasia and disorganization of the palatal shelf epithelium at E13.5. Scale bar: 20 µm. H.. Palatal explant culture assay reveals that mutant palatal shelves fuse appropriately. Palatal shelves freshly isolated at E13.5 were placed in contact and cultured for 48 h. ne, nasal epithelium; oe, oral epithelium. Download figure Download PowerPoint To test the possibility that the smaller size of the palatal shelves may result from reduced cell proliferation, we measured 5-bromodeoxyuridine (BrdU) incorporation between E13 and E13.5, the narrow time window during which most of the palatal shelf outgrowth occurs. At E13, cell proliferation was reduced significantly in the mutant epithelium and to a lesser extent in the mesenchyme (Fig 2E and F). At E13.5, BrdU incorporation was unaffected in the mesenchyme, but was still reduced significantly in the epithelium (Fig 2F). Consistent with the reduced proliferation observed, the p63+/L514F palatal epithelium appeared disorganized and hypoplastic with irregularly spaced basal cells expressing p63 and keratin 14 (Krt14). Basal cells were intermingled with Krt17- and Krt8-positive periderm cells, which lacked the characteristic flattened morphology observed in wild-type mice (Fig 2G). To test whether the disorganization of the epithelium could affect palatal fusion, we placed E13.5 palatal shelves with their medial edge epithelia in contact in culture. All explants fused appropriately (Fig 2H; n = 9), suggesting that the cleft palate in mutant mice is unlikely to be caused by a fusion failure. Thus, in p63+/L514 embryos cleft palate is caused by reduced cell proliferation as well as disorganization of the p63-expressing epithelium and consequent reduction of mesenchymal cell proliferation with insufficient extension of the palatal shelves toward the midline. Epidermal hypoplasia and defective cell proliferation in p63+/L514F embryos Severe hypoplasia was also observed in p63+/L514F epidermis (40% thinner than wild-type) during embryonic development and at birth, with a reduction in the number of nuclei and a more flattened appearance of cells in all layers (Fig 3A and B). In contrast, no hypoplasia was observed in epidermis of mice heterozygous for a null mutation in p63 (p63+/−) (Supporting Information Fig S4A). Figure 3. p63+/L514F epidermis is hypoplastic compared to the control. A.. H&E staining reveals hypoplasia of p63+/L514F epidermis at the indicated times of development. Dashed lines indicate the border between epidermis (top) and dermis (bottom). Scale bar: 50 µm. B.. Quantification of epidermal thickness (µm) in +/+ and p63+/L514F skin at birth (*p-value = 0.0009; n = 6). Data are represented as mean ± SD. C.. Immunofluorescence analysis for p63, Krt14 (basal layer), Krt10 (spinous layer), and loricrin (Lor, granular layer) at P0 reveals appropriate expression of these differentiation markers in mutant epidermis. Scale bar: 20 µm. D.. Immunoblotting of total cell extracts from neonatal epidermis using antibodies against the indicated differentiation markers, Krt1 (spinous layer), the upper spinous layer marker involucrin (Ivl), the adherens junction component E-cadherin (Cdh1) and αTubulin (Tub) as loading control. E.. BrdU staining of the epidermis reveals reduced proliferation in mutant epidermis during development. Left panels: BrdU positive cells are in red; nuclei are stained with DAPI. Dashed lines indicate the border between epidermis (top) and dermis (bottom). Scale bar: 50 µm. Right panel: BrdU positive cells are expressed as percentage of the total number of basal cells (E13.5 *p-value = 0.0151; n = 7. E16.5 *p-value = 0.0271; n = 7). Data are represented as mean ± SD. F.. Left panel: immunofluorescence for active caspase 3 at P0 showing low levels of apoptosis in p63+/L514F epidermis compared to wild-type. Arrows indicate apoptotic cells. Scale bar: 70 µm. Right panel: quantification of active caspase 3 positive cells. Data are represented as mean ± SD (p-value = 0.0010; n = 16). Download figure Download PowerPoint Differentiation markers were localized appropriately in neonatal and in developing mutant epidermis although each layer was hypoplastic compared to wild-type (Fig 3C; Supporting Information Fig S4B). Expression of these markers was quantitatively similar in wild-type and in mutant epidermis when cell extracts normalized for protein content (Fig 3D). Consistent with a normal differentiation process, the impermeable epidermal barrier was established at the appropriate developmental time-point in p63+/L514F embryos (Supporting Information Fig S4C), despite the epidermal hypoplasia. To test whether epidermal hypoplasia could be due to defective cell proliferation, we performed in vivo BrdU labelling at different embryonic stages. A statistically significant reduction in BrdU incorporation was detected in mutant epidermis at both E13.5 and E16.5 (Fig 3E). In contrast, at E18.5 the rate of BrdU incorporation was similar in mutant and wild-type epidermis in spite of persistent epidermal hypoplasia (Fig 3E). Apoptosis was a very rare event in p63+/L514F epidermis as determined by staining for active caspase 3 at E16.5 and P0 (Fig 3F and Supporting Information Fig S5A) or by fluorescent activated cell sorting (FACS) analysis with annexin V antibodies of freshly isolated keratinocytes (Supporting Information Fig S5B). The apoptotic rate was modestly increased in mutant versus wild-type epidermis. Thus, in p63+/L514F epidermis, hypoplasia is associated with a transient reduction of cell proliferation during embryonic development and a modest increase in apoptosis, whereas terminal differentiation is not significantly affected. Fgfr2b and Fgfr3b expression is downregulated in p63+/L514F mice To gain insight into the molecular mechanisms underlying the proliferative defects observed in the developing palate and skin, we compared gene expression profiles of mutant and wild-type cells or tissues derived from E14.5 skin, E18.5 epidermis and newborn primary keratinocytes. These data were also compared with previously obtained gene expression profiles of p63 knockdown versus control keratinocytes (Della Gatta et al, 2008). Several known p63 target genes were affected to a similar extent in p63+/L514F cells or tissue and in p63 knockdown keratinocytes compared to the appropriate controls (Supporting Information Table S1). However, the expression of selected cell cycle regulators that have been previously implicated in p63-mediated cell cycle progression, including p21Cip1/Waf1 (Nguyen et al, 2006; Truong et al, 2006), p16Ink4a, p19Arf (Su et al, 2009) and the micro-RNA miR-34a (Antonini et al, 2010), was unaffected in E14.5 mutant skin or in primary keratinocytes (Supporting Information Fig S6A), suggesting that reduced proliferation in mutant epidermis was unlikely to depend on their direct regulation. FGF signalling is a prominent pathway that controls epithelial cell proliferation during embryonic development. Fgfr2b−/− mice, which lack the epithelial-specific Fgfr2b isoform, display epidermal hypoplasia associated with a transient reduction of cell proliferation during development, tooth and hair defects and cleft palate with reduced palatal epithelial cell proliferation (De Moerlooze et al, 2000; Petiot et al, 2003; Rice et al, 2004). Interestingly, Fgfr2b expression is reduced in the developing epidermis and in thymus of p63−/− mice (Candi et al, 2007; Fomenkov et al, 2003; Laurikkala et al, 2006) and is affected by mutant p63 and by p63 knockdown in the global gene expression analysis (Supporting Information Table S1). To provide further support for Fgfr2b or other FGF signalling components being affected in p63+/L514F embryos, we firs" @default.
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• W1812133033 title "Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome" @default.
• W1812133033 cites W1482923899 @default.
• W1812133033 cites W1534987406 @default.
• W1812133033 cites W1930773806 @default.
• W1812133033 cites W1972366341 @default.
• W1812133033 cites W1978477306 @default.
• W1812133033 cites W1980943360 @default.
• W1812133033 cites W1986097125 @default.
• W1812133033 cites W1986285972 @default.
• W1812133033 cites W1989699495 @default.
• W1812133033 cites W1996094589 @default.
• W1812133033 cites W1998479150 @default.
• W1812133033 cites W1999261254 @default.
• W1812133033 cites W2007232912 @default.
• W1812133033 cites W2010807493 @default.
• W1812133033 cites W2014938226 @default.
• W1812133033 cites W2015555645 @default.
• W1812133033 cites W2017019345 @default.
• W1812133033 cites W2018212036 @default.
• W1812133033 cites W2018528375 @default.
• W1812133033 cites W2023332224 @default.
• W1812133033 cites W2026863640 @default.
• W1812133033 cites W2042239273 @default.
• W1812133033 cites W2044672520 @default.
• W1812133033 cites W2051113417 @default.
• W1812133033 cites W2054052732 @default.
• W1812133033 cites W2056838445 @default.
• W1812133033 cites W2057586248 @default.
• W1812133033 cites W2058991912 @default.
• W1812133033 cites W2063603189 @default.
• W1812133033 cites W2075430446 @default.
• W1812133033 cites W2075799212 @default.
• W1812133033 cites W2080849609 @default.
• W1812133033 cites W2081375866 @default.
• W1812133033 cites W2081852205 @default.
• W1812133033 cites W2086879086 @default.
• W1812133033 cites W2087340322 @default.
• W1812133033 cites W2089631233 @default.
• W1812133033 cites W2100465544 @default.
• W1812133033 cites W2116358704 @default.
• W1812133033 cites W2116718098 @default.
• W1812133033 cites W2129246982 @default.
• W1812133033 cites W2129684968 @default.
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• W1812133033 cites W2156378083 @default.
• W1812133033 cites W2157209768 @default.
• W1812133033 cites W2158690038 @default.
• W1812133033 cites W2161092951 @default.
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