Generation and validation of ΔNp63 knockout mice

The p63 gene is transcribed using two alternative promoters, generating TAp63 transcripts from an upstream (P1) promoter and ΔNp63 variants that are transcribed using the downstream promoter (P2) embedded within intron 3 (supplementary material Fig. S1A). Translation of the ΔNp63 protein isoforms is initiated at an AUG codon located in exon 3′, an exon that is unique to the ΔNp63 isoform (supplementary material Fig. S1A). To target ΔNp63 while ensuring the TAp63 isoforms remain unaffected, the coding sequence of the ΔNp63-specific exon was replaced by GFP (supplementary material Fig. S1B). This knock-in strategy also allowed for expression of GFP in place of ΔNp63 while under the influence of the endogenous ΔNp63 regulatory control elements. ΔNp63^gfp/+ ES cells were generated by homologous recombination and subsequently used to derive mice that were either heterozygous or homozygous for this mutation (supplementary material Fig. S1C). Importantly, using RT-PCR we showed that the ΔNp63 mRNA was absent in the homozygous mutant mice, whereas TAp63 mRNA transcripts remain unaffected, demonstrating selective inactivation of the ΔNp63 isoforms (supplementary material Fig. S1D). Loss of ΔNp63 was further verified by performing immunofluorescence on whole embryo tissue sections from E14.5 ΔNp63^gfp/gfp mice using a ΔNp63-specific antibody (supplementary material Fig. S1E, top). Moreover, immunostaining on sections of ΔNp63^gfp/gfp ovaries with anti-TAp63 antibodies showed robust TAp63 expression in the follicles, consistent with previous reports demonstrating high levels of this isoform in ovaries and confirming that the expression of TAp63 proteins is not disrupted in these animals (supplementary material Fig. S1E, bottom) (Suh et al., 2006).

In order to confirm that the GFP expression mirrored that of endogenous ΔNp63, we performed immunofluorescence staining on various epithelial tissues from heterozygous ΔNp63^gfp/+ animals. Examination of dorsal skin samples from postnatal day 7 ΔNp63^gfp/+ mice clearly revealed that GFP expression colocalized with ΔNp63. More specifically, GFP was expressed in the basal layer of the epidermis and outer root sheath of the hair follicles, which have been shown to express high levels of ΔNp63 (supplementary material Fig. S2). Similar colocalization of GFP and ΔNp63 was also detected in oral tissues, such as the buccal mucosa and the dorsal surface of the tongue, in ΔNp63^gfp/+ mice (supplementary material Fig. S2).

ΔNp63 mutant animals phenocopy mice lacking all p63 isoforms and fail to develop a normal stratified epidermis

Upon intercrossing of ΔNp63^gfp/+ animals, all resulting ΔNp63^gfp/gfp null mice died shortly after birth, presumably owing to severe developmental defects and dehydration. In order to investigate the loss of ΔNp63 in epithelial morphogenesis, embryos were harvested at various stages of development beginning at E14.5. Heterozygous ΔNp63^gfp/+ mice taken at all time points examined appeared indistinguishable from wild-type littermates. By contrast, ΔNp63^gfp/gfp mice were smaller than their wild-type counterparts and exhibited obvious defects in limb and craniofacial development and a lack of epidermis that resulted in an overall shiny, translucent appearance (Fig. 1, left). Of the 40 homozygous mutant embryos examined so far, all animals exhibited arrested hind limb and forelimb development, albeit with some degree of variability in the extent of defects (supplementary material Fig. S3). Closer examination of paraffin-embedded sections of whole embryos revealed a failure of keratinocytes to develop into a mature stratified epidermis in the ΔNp63^gfp/gfp animals as compared with wild-type mice (Fig. 1, middle). Interestingly, at E14.5, ΔNp63-null embryonic skin consisted of a thin layer of intact epithelial cells, which appeared hypoplastic (∼2 cell layers in thickness) and disorganized. By contrast, control E14.5 wild-type skin was typically 3-4 cell layers thick. This disorganized pattern of epithelial keratinocytes persisted at E16.5, along with obvious defects in epidermal organization. By E18.5, ΔNp63^gfp/gfp surface keratinocytes were present in sparse clumps, which covered portions of the exposed lower dermis and failed to show overt signs of normal epidermal stratification and maturation, as surface keratinocytes remained 1-2 cell layers in thickness in most of these areas (Fig. 1, middle). Moreover, there were no signs of hair follicle and sebaceous gland morphogenesis in the ΔNp63 knockout animals at any of the embryonic stages examined.

Given the strong expression pattern of p63 in the basal cells of many stratified epithelial tissues, we next examined the effects of the loss of ΔNp63 in the oral cavity, a tissue with high levels of ΔNp63 expression. Similar to what we observed in the skin, the tongue, palate and the buccal mucosa of E18.5 ΔNp63-null animals lacked a mature stratified epithelium, confirming an essential role for this p63 isoform in the formation of these stratified epithelial tissues (Fig. 1, right). In general, judging by morphological and histological analysis, the ΔNp63^gfp/gfp animals appear to largely phenocopy the epithelial aplastic phenotype of the previously reported pan-p63-null animals (Mills et al., 1999; Yang et al., 1999).

ΔNp63-null keratinocytes undergo accelerated differentiation, as evident by the premature developmental expression of terminal differentiation markers

To better define the specific role of ΔNp63 during embryonic skin development and to help resolve the lingering questions regarding the overall cellular nature of the phenotypic changes that result from the loss of p63, we next examined the ΔNp63^gfp/gfp skin in greater detail using a battery of established markers. To investigate whether cells of the surface ectoderm of ΔNp63^gfp/gfp animals were able to commit to an epidermal cell lineage, we analyzed skin sections at E13.5, a stage at which this process is already initiated. As expected, we identified robust expression of K5 in the epidermis of control animals, with expression of K8, a marker of the simple epithelium, being completely absent (Fig. 2, left). This was true for all the subsequent developmental time points that we examined in the control animals. In the ΔNp63^gfp/gfp mice, keratinocytes that covered the surface of the embryo also expressed K5, albeit to a weaker extent and in a patchy fashion compared with control animals. These data confirm the ability of at least some cells lacking ΔNp63 to commit to an epidermal cell fate. Interestingly, we also identified clusters of keratinocytes that, although they expressed K5, also retained expression of K8 (Fig. 2, middle). This was particularly prominent in anatomical areas that are normally protected from abrasion and friction, such as the ventral neck and chest. In some areas that were more exposed, such as the dorsal back and head regions, we found a single layer of cells expressing K8 (Fig. 2, right). These K8-retaining epithelial cells were present at multiple stages of embryonic development. Together, these data suggest that, in the absence of ΔNp63, keratinocytes in at least some areas of the embryonic skin are able to commit to an epidermal lineage. However, there also appears to be an intermediate state, suggesting a developmental conundrum in which the epithelial cells simply fail to commit or co-express simple epithelial (K8) and basal keratinocyte (K5) specific markers.

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Fig. 2.

Dorsal and ventral skin sections of E13.5-18.5 wild-type and ΔNp63^gfp/gfp animals stained for K5 and K8. Antibody staining for K5 (red) and K8 (green) reveals that epidermal keratinocytes in the ΔNp63^gfp/gfp mice express the simple epithelial marker K8, which is absent in the wild-type animals (compare right and left panels). Some ΔNp63^gfp/gfp keratinocytes co-express K5 and K8 (middle panel, yellow staining). Scale bars: 37.5 μm.

Given the ability of the keratinocytes of ΔNp63^gfp/gfp mice to commit to an epidermal cell fate, as demonstrated by the expression of K5 and K14 (Fig. 2; data not shown), we sought to further confirm this observation by investigating the status of the transcription factor AP-2α (Tfap2a – Mouse Genome Informatics), which is highly expressed in basal keratinocytes and is important for basal gene expression (Maytin et al., 1999; Sinha et al., 2000; Wang et al., 2006). Robust expression of AP-2α was evident in both the wild-type and ΔNp63^gfp/gfp skin samples (supplementary material Fig. S4), verifying that, in the absence of this specific p63 isoform, keratinocytes can form a basal layer and express some of its distinctive markers.

After confirming formation of the basal layer in the absence of ΔNp63, we next sought to investigate whether the cells were able to progress through to subsequent stages of keratinocyte differentiation. We co-stained ΔNp63^+/+ skin sections for K5 and K1 or K10, which allowed us to simultaneously mark the basal (K5) and spinous (K1, K10) layers. As expected, we observed a clear separation between these layers at E15.5 in control animals (supplementary material Fig. S5). By contrast, keratinocytes in several areas of ΔNp63^gfp/gfp skin exhibited co-expression of K5 with K1 and K10, suggesting an altered differentiation state. Although the boundary between the basal and spinous layer was blurred in ΔNp63-null skin, the keratinocytes were able to initiate the formation of a multilayered stratified epithelium, as evident by small pockets of cells that were stacked on top of each other (Fig. 3; supplementary material Fig. S5). The formation of the spinous layer in ΔNp63^gfp/gfp keratinocytes was further confirmed by the expression of the transcription factor Gata3, which normally marks this layer (de Guzman Strong et al., 2006; Kaufman et al., 2003; Kurek et al., 2007) (supplementary material Fig. S4, bottom). Interestingly, we also observed the expression of markers associated with terminal differentiation, including loricrin, filaggrin and involucrin in the ΔNp63-null E15.5 embryos. Such expression at this early developmental window was notably premature given that these markers were conspicuously absent in the counterpart wild-type control littermate embryos (Fig. 3). We confirmed these results by quantifying the expression levels of several of these markers of differentiation by qRT-PCR at E14.5 and E15.5 (supplementary material Fig. S6A).

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Fig. 3.

Accelerated keratinocyte differentiation in the absence of ΔNp63. Dorsal skin sections of E15.5 wild-type and ΔNp63^gfp/gfp mice stained for various markers associated with the keratinocyte stratification (K5) and differentiation (K10) program reveal that ΔNp63-null keratinocytes are able to progress through each program. Examination of markers associated with late differentiation (Lor, loricrin; Fil, filaggrin; Inv, involucrin) reveal that ΔNp63-null keratinocytes undergo premature terminal differentiation as compared with wild-type keratinocytes. Nuclei are stained with DAPI. Scale bars: 37.5 μm.

Given that such a precocious differentiation program of embryonic p63-null keratinocytes had not been reported previously, we examined the status of the markers of late differentiation in the Brdm2 mouse model, which lacks all isoforms of p63 (Mills et al., 1999). We were unable to detect the expression of any of the late markers of differentiation at E15.5 in the Brdm2 mouse (supplementary material Fig. S7), a feature that was consistently evident in our ΔNp63-null animals. This result suggests that there are functionally distinct outcomes in these two mouse models, which could reflect the effects of remnant p63 levels, isoform-specific activities or differences in strain background. Importantly, we demonstrate that, although keratinocytes are able to undergo terminal differentiation in the absence of ΔNp63, this process appears unbalanced and accelerated.

ΔNp63 maintains the proliferative potential of basal keratinocytes in embryonic epidermis

During the early stages of skin morphogenesis, epidermal keratinocytes undergo massive proliferation in order to maintain adequate cell coverage of the quickly growing embryo. Since only small patches of the epidermis were present in the skin surface of the ΔNp63^gfp/gfp embryos, we wondered whether the proliferative potential of the epidermis was compromised. We examined ΔNp63^gfp/gfp skin samples for expression of the proliferation marker Ki67 from two distinct stages when the cells are normally undergoing rapid proliferation (E13.5 and E16.5). Although both the ΔNp63^gfp/gfp and wild-type epidermis showed Ki67-positive nuclear staining, a dramatic reduction in Ki67 expression was evident in the ΔNp63^gfp/gfp skin (quantified in supplementary material Fig. S8A,B, top and middle). These data thus demonstrate that the proliferative potential of keratinocytes is reduced in the absence of ΔNp63, which is consistent with previous knockdown studies in cell culture (Truong et al., 2006).

Given the hypoplastic nature of the ΔNp63^gfp/gfp skin, we wondered whether this phenotype was also affected by alterations in the rate of cellular apoptosis. Immunostaining with an antibody recognizing cleaved caspase 3 revealed no evidence of enhanced apoptosis in the skin of ΔNp63 knockout animals at E13.5 or E16.5 (supplementary material Fig. S8A,B, bottom).

Loss of components of the extracellular matrix and cellular junctions in the absence of ΔNp63

One of the key components necessary for proper epithelial formation and integrity is the proper formation of the extracellular matrix (ECM)-rich basement membrane (Fuchs and Raghavan, 2002). The basement membrane is generated from the cells of the basal layer, which are responsible for the production, secretion and assembly of this important structure. Given the lack of a mature stratified epidermis in our mutants, we wondered whether there was a defect in the formation of the basement membrane in these animals. We examined the expression of a panel of protein markers important in the formation of the basement membrane. Interestingly, we found several prominent basal ECM proteins, including laminin α5 and collagen IV, to be severely reduced in the ΔNp63^gfp/gfp mice as compared with control animals (Fig. 4). We also found reduced levels of nidogen, heparan sulfate proteoglycan core protein (HSPG; Hspg2 – Mouse Genome Informatics), β1-integrin and E-cadherin in the mutant animals as compared with control littermates (Fig. 4). Importantly, similar results were obtained with sections from both the dorsal and ventral surfaces of the skin (data not shown) in the ΔNp63^gfp/gfp animals, as compared with the controls, suggesting no differences between these anatomical regions. A quantification of the mRNA expression levels for a subset of these ECM genes (supplementary material Fig. S6B) matched the corresponding protein expression profiles.

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Fig. 4.

Loss of key components of the extracellular matrix in the epidermis of ΔNp63-null mice. Dorsal skin sections from E15.5 ΔNp63^gfp/gfp mice reveal a dramatic reduction in several crucial basement membrane proteins, including laminin α5 (Lamin5) and collagen IV (COLIV), with modest to moderate changes in E-cadherin (E-Cad), HSPG, nidogen and integrin β1 (β1) expression. Sections are co-stained for K5 as indicated and nuclei are counterstained with DAPI. Scale bars: 37.5 μm.

In addition to the ECM, cell-cell adhesion junctions play an important role in maintaining epidermal integrity and homeostasis (Green et al., 2010). Given the disorganized appearance of the epidermis of the ΔNp63^gfp/gfp animals, we investigated the status of hemidesmosome and desmosome junctions, which are crucial components of the cell-substrate and cell-cell adhesion machinery, respectively, in the skin. We found reduced staining for integrins α6 and β4 in both the dorsal and ventral surfaces of the ΔNp63^gfp/gfp skin, suggesting that hemidesmosomes were compromised (Fig. 5). We next examined the expression profiles of several desmosomal components, including plakophilin 2, which is normally expressed in the basal layer, and desmoglein 1 and desmoglein 2, which are expressed in the suprabasal layers (Kottke et al., 2006). As shown in Fig. 5, at E15.5 we discovered a dramatic reduction in desmoglein 1 expression and moderate reductions in desmoglein 2 in both the dorsal and ventral surfaces of the skin of ΔNp63^gfp/gfp animals as compared with control mice. However, we did not observe appreciable differences in plakophilin 2 staining between ΔNp63^gfp/gfp and wild-type skin. These results were further substantiated by examining the corresponding mRNA levels by qRT-PCR (supplementary material Fig. S6C). Taken together, our data indicate that in the absence of ΔNp63 there is a generalized failure of keratinocytes to properly form the ECM, desmosome and hemidesmosome junctions. It is likely that these broad defects, in combination with the reduction in cellular proliferation, contribute to the loss of epithelial cell integrity and impaired skin development observed in our ΔNp63 mutant animals.

Loss of ΔNp63 is associated with alterations in the Notch signaling pathway

We next asked whether the epidermal differentiation phenotype observed in the ΔNp63^gfp/gfp animals is associated with changes in Notch signaling given its importance in mammalian epidermal differentiation and the well-established molecular link between p63 and Notch (Blanpain et al., 2006; Laurikkala et al., 2006; Nguyen et al., 2006; Watt et al., 2008; Williams et al., 2011). Furthermore, an accelerated epidermal differentiation phenotype similar to that observed in the E15.5 ΔNp63^gfp/gfp skin has been reported in animals with targeted deletion of Hes1, a crucial Notch effector (Moriyama et al., 2008). We began our initial investigation by examining the gene expression levels of various components of the Notch signaling pathway by qRT-PCR at E14.5 and E15.5 in ΔNp63^gfp/gfp and control embryos (supplementary material Fig. S6D). In the absence of ΔNp63, the expression of several components of the Notch signaling pathway was reduced, including Hes1, Jag1 and Notch1, a trend that was particularly evident in E15.5 embryos. Interestingly, several of these genes have previously been identified as direct transcriptional targets of p63 (Dotto, 2009; Nguyen et al., 2006; Sasaki et al., 2002; Yang et al., 2006). However, to our surprise, we also observed elevated levels of Notch2, Notch3 and Rbpj mRNAs in ΔNp63^gfp/gfp embryos, suggesting that the loss of ΔNp63 has opposing effects on the expression of individual Notch receptors and related genes within this pathway. To examine whether these changes occurred specifically in the skin epidermis, we performed immunofluorescence staining on dorsal skin sections of E15.5 control and ΔNp63^gfp/gfp mice. Interestingly, we found reduced levels of Hes1 and Notch1 in the ΔNp63^gfp/gfp skin sections, whereas suprabasal Notch2 and Notch3 staining was stronger than in control animals (Fig. 6). These data confirm our qRT-PCR observations and reveal that the loss of ΔNp63 results in altered expression levels of various components of the Notch signaling pathway.

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Fig. 5.

Loss of ΔNp63 affects the expression of hemidesmosome and desmosome junction proteins. Immunofluorescence staining of dorsal and ventral skin sections at E15.5 reveals reduced expression of the hemidesmosomal components integrin α6 and β4 in ΔNp63^gfp/gfp mice. Alterations to various desmosome junction proteins are also seen. All sections are co-stained for K5 and counterstained with DAPI. Dsg1, desmoglein 1; Dsg2, desmoglein 2; Pkp2, plakophilin 2. Scale bars: 37.5 μm.

Whereas the relationship between p63 and Notch1 has been well established by prior studies, the link between p63 and Notch2 and Notch3 has not received much attention, particularly in the context of keratinocyte development and differentiation (Nguyen et al., 2006). The elevated levels of Notch2 and Notch3 in ΔNp63^gfp/gfp animals prompted us to carefully investigate genomic data sets generated by the ENCODE project (Myers et al., 2011) and p63 ChIP-seq experiments performed with human keratinocytes (Kouwenhoven et al., 2010). We specifically focused on regulatory regions that were in an open chromatin confirmation and contained histone modification signatures associated with regulatory elements. More importantly, because no such data are currently available from mouse, we also focused on regulatory regions that were evolutionarily conserved between human and mouse. Whereas none of the potential regulatory elements surrounding the Notch2 gene met such strict criteria (data not shown), one region within intron 2 of Notch3, as identified by ChIP-seq experiments, was worthy of closer examination (Fig. 7A). This region in normal human epidermal keratinocytes (NHEKs) is likely to be in an open chromatin conformation as suggested by DNase-seq data. Concomitantly, there is also enrichment for chromatin modification marks that are typically associated with enhancers [H3K4me1 and H3K4me2 (Barski et al., 2007; Bernstein et al., 2005; Birney et al., 2007; Heintzman et al., 2007)] and active regulatory regions [H3K9ac and H3K27ac (Bernstein et al., 2005; Heintzman et al., 2009)] (Fig. 7A). Closer examination of this DNA region revealed high sequence conservation between human and mouse, as shown by the Genome Vista alignment (Fig. 7B) and the presence of two closely spaced potential p63 binding motifs (data not shown). To test whether p63 can bind to the corresponding site of the mouse Notch3 gene, we performed ChIP followed by qPCR experiments with cross-linked DNA isolated from mouse keratinocytes grown in culture. p63-bound chromatin was immunoprecipitated with an anti-ΔNp63 antibody [RR14 (Romano et al., 2006)] and an anti-p63 antibody that recognizes the alpha isoforms of p63 (H129). Quantitative PCR was then performed with primers spanning the p63 binding sites within this region. In both of the p63 immunoprecipitations, we found strong enrichment of the putative p63 response segment in the Notch3 gene relative to the IgG control after normalization to a random genomic locus (Fig. 7C). These data strongly suggest direct binding of ΔNp63 to a critical regulatory region within intron 2 of Notch3.

We extended our studies to the murine Rbpj gene because it was consistently upregulated in our qRT-PCR analysis of ΔNp63^gfp/gfp animals. One evolutionarily conserved element was identified in a region 3′ of Rbpj, which was marked with distinctive chromatin states typical of regulatory elements (supplementary material Fig. S9A). ChIP experiments performed in mouse keratinocytes using the RR14 and H129 antibodies revealed specific enrichment of this region, providing evidence of ΔNp63 directly regulating this gene (supplementary material Fig. S9B).

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Fig. 6.

Effects of ΔNp63 loss on components of the Notch signaling pathway. Dorsal skin sections at E15.5 reveal increased expression levels of Notch2 and Notch3, whereas Hes1 and Notch1 appear modestly reduced, in ΔNp63^gfp/gfp compared with wild-type mice. Insets show higher magnification views. Sections are co-stained for K5 and counterstained with DAPI. Scale bars: 37.5 μm.

Collectively, our studies, coupled with those from prior investigations, support the notion that p63 directly regulates various components of the Notch signaling pathway and suggest that these cumulative effects of altered Notch signaling in part contribute to the accelerated keratinocyte differentiation phenotype observed in ΔNp63^gfp/gfp animals.