Epidermal development

The signaling cascades responsible for the onset of epidermal development are poorly understood, but are likely to involve cross-talk between the ectoderm and the underlying mesen-chyme. The molecules mediating this cross-talk, however, are currently unknown. The single-layered ectoderm covering the body expresses keratin 8 (K8) and K18 (^{Jackson et al, 1981}). At embryonic day 9.5 (E9.5), prior to the onset of stratification, this simple epithelium initiates expression of K5, K14, and p63 (^{Byrne et al, 1994;Mills et al, 1999;Yang et al, 1999}), proteins expressed in the basal layer of adult epidermis. Shortly after, at E10.5, the first stage of epidermal differentiation results in the formation of the periderm (^{M'Boneko and Merker, 1988}). Further epidermal maturation takes place between E15.5 and E18.5. At E15.5, the epidermis forms an intermediate layer between the basal layer and the periderm, which corresponds to the adult spinous layer and is marked by the onset of K1 and K10 expression (^{Bickenbach et al, 1995}). Loricrin and filaggrin are first expressed between E16.5 and E17.5 as the granular layer and stratum corneum form, creating a water impermeable barrier (^{Rothnagel et al, 1987;Mehrel et al, 1990;Bickenbach et al, 1995}). At E18.5 the epidermis is fully stratified and the periderm is shed.

p63 in epidermal development

Initiation of epidermal development at E9.5 coincides with the onset of p63 expression in the single-layered surface ectoderm, suggesting a role for p63 in early epidermal development. The highest levels of p63 expression are observed in the apical ectodermal ridge (AER), a specialized, pseudo-stratified epithelial structure required for limb bud outgrowth (^{Johnson and Tabin, 1997;Mills et al, 1999;Yang et al, 1999}). After E9.5, p63 continues to be expressed in the basal layer of the developing and adult epidermis and other stratified and pseudo-stratified epithelia.

The importance of p63 in ectodermal development is underscored by the severe developmental defects observed in p63^-/- mice (^{Mills et al, 1999;Yang et al, 1999}). Newborn mice lacking functional p63 do not have stratified and pseudo-stratified epithelia, lack epithelial appendages such as mammary glands, hair follicles, and teeth, have truncated forelimbs, and do not have hindlimbs. At birth, p63^-/- mice do not have a recognizable epidermis but possess a thin, single-cell layer covering the body, resulting in dehydration and death within hours after birth. This cell layer fails to express numerous epidermal differentiation markers including K5, K14, K1, K10, loricrin, filaggrin, and involucrin, indicating a fundamental defect in epithelial lineage development. As appendage development requires signaling between the ectoderm and the underlying mesenchyme, the defects in appendage development in p63^-/- mice may be secondary to the failure to develop stratified epithelial structures, such as the epidermis and the AER. Indeed, the expression patterns of various genes required for limb bud outgrowth suggest that improper AER formation in p63^-/- mice results in a failure of the ectoderm to participate in ectodermal-mesenchymal signaling. This, in turn, probably underlies the defects in limb morphogenesis observed in p63^-/- mice.

This defect in epithelial lineage development has led to two, not mutually exclusive, hypotheses concerning the potential role of p63 in epidermal development. Patches of cells expressing the terminal differentiation markers loricrin, filaggrin, and involucrin were observed in the epidermis of E17.5 p63^-/- embryos generated by Yang et al, suggesting that the embryonic p63^-/- epidermis undergoes a process of differentiation before undergoing cell death (^{Yang et al, 1999}). This observation led Yang et al to suggest that p63 may play a pivotal role in maintaining the epidermal stem cell population. Thus, the absence of squamous epithelia in p63^-/- mice could be explained by a premature depletion of the stem cell compartment resulting in a failure to maintain stratified epithelia. This hypothesis was strengthened by clonal analysis of wild-type keratinocytes that demonstrated exclusive expression of p63 in holoclones, thought to represent the stem cell compartment of the epidermis (^{Barrandon and Green, 1987;Pellegrini et al, 2001}). Although these results are consistent with a role for p63 in stem cell maintenance, definitive evidence has not been reported. An attractive alternative explanation for the absence of squamous epithelia in p63^-/- mice is that p63 plays a critical role in the commitment of embryonic ectoderm to squamous epithelial lineages. In fact, although patches of differentiated cells were observed by Yang et al (1999), we have been unable to detect such patches at any stage during embryonic development of p63^-/- mice (^{Mills et al, 1999}). In addition, recent evidence suggests that p63 regulates the expression of several genes potentially involved in epidermal development and differentiation (^{Dohn et al, 2001;Nishi et al, 2001;Sasaki et al, 2001}). Therefore, a plausible role for p63 is the induction of a squamous differentiation program.

Structure of p63

The understanding of the precise function of p63 in epithelial development is complicated by the finding that p63 is transcribed into at least six different isoforms generated by alternative promoter usage and alternative splicing (Figure 1) (^{Yang et al, 1998}). Alternative promoter use gives rise to two classes of p63 isoforms, those containing an acidic amino terminus analogous to the p53 transactivation domain (TA isoforms) and those lacking this domain (N isoforms). In addition, alternative splicing gives rise to three different carboxy termini designated , , and . The carboxy terminus is the longest and contains a sterile motif (SAM) domain (^{Chi et al, 1999}). SAM domains are protein interaction motifs frequently found in proteins involved in development and differentiation, suggesting a role for p63 isoforms in these processes.

Figure 1.

Structure of p63 isoforms. Alternative promoter usage gives rise to p63 isoforms containing a transactivation domain and isoforms lacking this domain. Three carboxy termini are generated by alternative splicing. Exons are color coded indicating the functional domains. Adapted from Yang et al (2000).

Full figure and legend (24K)

p63 target genes

In vitro studies demonstrated that p63 is able to bind to the p53 response element (^{Bian and Sun, 1997;Zeng et al, 1998}) containing two copies of the 10-mer PuPuPuC(A/T)(T/A)GPyPyPy separated by two to eight nucleotides (^{el Deiry et al, 1992}). The p53 response element in promoters of different p53 target genes can contain as many as four mismatches. Interestingly, biochemical studies on binding of p63 to p53 response elements of a subset of p53 target genes demonstrated that, as opposed to p53, p63 has a higher affinity for p53 response elements that contain more mismatched nucleotides (e.g., Bax-1), suggesting that p53 and p63 may have overlapping as well as different transcriptional targets (^{Zeng et al, 1998}).

In addition to being able to bind to p53 response elements, transient transactivation assays demonstrated that p63 can induce transcription of several p53 responsive genes. As predicted from their structures, these studies suggested that only TA isoforms are capable of transactivating p53 target genes, whereas N isoforms have a dominant-negative function (^{Yang et al, 1998}). In addition, functional consequences of carboxy terminal variability have been described. Most notably, TAp63, which contains a transactivation domain, is unable to transactivate p53 target genes in transient transactivation assays. Although these experiments provide valuable insight into the mechanism of action of p63, it is well established that the activation of transfected promoter constructs does not always reflect the activation of endogenous genes where chromatin remodeling occurs (^{Smith and Hager, 1997}. In this context, it is of interest to note that recent transfection studies have identified target genes of p63 that are potentially involved in epidermal differentiation and development.

TAp63 was shown to induce expression of EphA2 tyrosine kinase, which inhibits integrin-mediated cell adhesion, potentially contributing to epidermal differentiation (^{Miao et al, 2000;Dohn et al, 2001}). In addition, TAp63 was shown to induce expression of Jagged-1 and also to downregulate epidermal growth factor receptor (EGFR) expression (^{Nishi et al, 2001;Sasaki et al, 2001}). Jagged-1, a Notch ligand, activates Notch signaling, which is thought to promote epidermal development (^{Powell et al, 1998;Lowell et al, 2000}). Downregulation of EGFR expression is associated with epidermal differentiation as demonstrated by the sharp decrease of EGFR expression levels in the suprabasal layers compared to the basal layer of the epidermis (^{Zambruno et al, 1990}). In summary, these data demonstrate that p63 target genes are potentially involved in epidermal development and differentiation.

Role of p63 in human disease

It is well known that many developmental genes continue to play an important role in regulation of cell growth and differentiation after embryogenesis. The dysregulation of these genes has been associated with congenital abnormalities and cancer. Although mutations in p63 are rare in human cancers (^{Hagiwara et al, 1999}), dysregulated expression of p63, sometimes in conjunction with amplification of its genomic region at 3q27–28, is frequently observed in a subset of human epithelial cancers (^{Crook et al, 2000;Hibi et al, 2000;Park et al, 2000;Yamaguchi et al, 2000}). The isoform targeted for overexpression appears to be Np63, which, in this context, may downregulate expression of p53 target genes thereby preventing the induction of cell cycle arrest and apoptosis. Interestingly, we have demonstrated a reduction in ultraviolet-B-induced apoptosis in mice expressing Np63 under control of the mouse loricrin promoter suggesting that dominant-negative isoforms of p63 may play an oncogenic role in epithelial tissues (^{Liefer et al, 2000}).

Congenital abnormalities associated with p63 dysregulation include a subset of ectodermal dysplasias. Ectodermal dysplasias comprise a large and heterogeneous group of conditions characterized by hereditary, developmental disturbances that affect tissues of ectodermal origin. Mutations in p63 have been shown to underlie ectrodactyly, ectodermal dysplasia and cleft lip (EEC), limb–mammary syndrome (LMS), split hand-split foot malformation (SHFM), ankyloblepharon ectodermal dysplasia and clefting (AEC or Hay-Wells disease), and acro-dermato-ungual-lacrimal–tooth syndrome (ADULT). Each of these syndromes has been shown to result from different types of p63 mutations. Missense mutations in the DNA-binding domain of p63 result in EEC (^{Celli et al, 1999}) or ADULT syndrome (^{Duijf et al, 2002}), missense mutations in the SAM domain result in AEC (^{McGrath et al, 2001}), frame-shift mutations in or near the SAM result in LMS (^{van Bokhoven et al, 2001}), whereas p63 mutations in SHFM are dispersed throughout the gene (^{Ianakiev et al, 2000}). Expression constructs encoding individual p63 isoforms with each identified mutation were used for transactivation assays using a reporter gene under control of the p53 response element. Only subtle differences between AEC and EEC mutations with respect to transactivation were observed. Interestingly, the missense mutation observed in ADULT syndrome was demonstrated to result in a gain-of-function of the Np63 isoform (^{Duijf et al, 2002}). To gain a better understanding of the functional implications of these p63 mutations, it is imperative to understand the physiologic role of p63 in skin development.

In summary, the precise role of p63 in epidermal development is still elusive and may include stem cell maintenance and/or the induction of a squamous differentiation program. A first step towards the understanding of the role of p63 in epidermal development is to identify the isoform(s) responsible for this process. In addition, understanding the role of p63 will be greatly facilitated by the identification of p63 downstream genes as well as the proteins binding to p63. Comparisons between the target genes and protein-binding partners of wild-type and mutant p63 will provide insight into the molecular basis of ectodermal dysplasias and may identify therapeutic targets.

Location of epidermal stem cells

An equally important process required for epidermal development and differentiation is the regulation of epidermal stem cell fate. Recent progress has been made in identifying the molecules involved in stem cell fate decisions allowing for a better understanding of this process.

The characteristics of a stem cell include a high capacity for self-renewal throughout adult life and the ability to produce daughter cells that undergo terminal differentiation (^{Lajtha, 1979}). Stem cells have been found in permanently renewing tissues including the hemopoietic system, the small intestine lining, and the epidermis (^{Jones and Watt, 1993}). Epidermal stem cells give rise to transit amplifying cells, which have a high potential to undergo differentiation and a low potential for self-renewal compared to stem cells (^{Jones and Watt, 1993}). Epidermal stem cells have been shown to express high levels of ₁ integrin compared to transit amplifying cells. This has provided a way to isolate epidermal stem cells based on their adhesiveness to extracellular matrix proteins (^{Jones and Watt, 1993;Bickenbach and Chism, 1998}).

In the epidermis, cells residing in the bulge and bulb regions of hair follicles have characteristics of stem cells (^{Reynolds and Jahoda, 1991;Hardy, 1992}). Label retaining experiments demonstrated that cells residing in the bulge of mouse pelage follicles are slow cycling cells. The progeny of these slow cycling cells contribute to hair growth of mouse pelage follicles and play a role in closure of epidermal wounds (^{Taylor et al, 2000}). Additional evidence to suggest that epidermal stem cells reside in the bulge region of the hair follicles was provided by a series of elegant experiments performed by^{Oshima et al (2001)}. Chimeric hair follicles, created from a wild-type vibrissal follicle with an amputated bulge region and the bulge region from a Rosa 26 vibrissal follicle, were transplanted into athymic mice. Rosa 26 transgenic mice constitutively express a lacZ reporter gene (^{Friedrich and Soriano, 1991}). Therefore the Rosa 26 bulge region provided labeled cells whose migration could be monitored in the wild-type follicle. Four weeks after the transplant, labeled cells could be seen migrating along the hair follicle towards the hair bulb and the epidermis. After 6 weeks, labeled cells were found in the hair bulb, sebaceous glands, and epidermis (^{Oshima et al, 2001}). These data suggest that epidermal stem cells reside in the bulge region of hair follicles from where they migrate to generate hair follicles, sebaceous glands, and epidermis (Figure 2).

Figure 2.

Multipotent stem cells generate hair follicles, sebaceous glands, and the epidermis. Multipotent stem cells (red) residing in the bulge region of the hair follicle migrate (pink) towards the hair bulb region and epidermis, giving rise to differentiated cells (purple) that populate the hair follicle, sebaceous gland, and epidermis. Inset shows that, in the epidermis, stem cells (pink) give rise to transit amplifying cells (blue), which proliferate and then differentiate giving rise to progeny at progressive stages of maturation (multiple shades of green).

Full figure and legend (26K)

Wnt signaling regulates epidermal stem cell fate decisions

Although there are strong data suggesting the location of epidermal stem cells, we are just starting to determine how epidermal stem cell fate is regulated. There is evidence to suggest that proteins involved in the Wnt signaling pathway have a role in epidermal stem cell fate decisions (^{Gat et al, 1998;Kishimoto et al, 2000}). The Wnt signaling pathway is important for body axis formation and for the development of the central nervous system, limbs, and mammary glands. In addition, this pathway has been found to regulate the hair cycle (^{Kishimoto et al, 2000}). The Wnt signaling pathway is activated by the binding of a glycoprotein of the Wnt protein family to a receptor of the frizzled transmembrane protein family. A signal is transduced to dishevelled, a cytoplasmic protein, which in turn represses the activity of glycogen synthase kinase-3, thereby stabilizing -catenin. In the absence of Wnt signaling, phosphorylation of -catenin by glycogen synthase kinase-3 targets -catenin for degradation via ubiquitination. -Catenin, first identified as a cell-adhesion protein, is stabilized by Wnt signaling. After stabilization, -catenin translocates to the nucleus where it binds to coactivators from the Lef/TCF family and activates the transcription of target genes (reviewed in^{Polakis, 2000}). In the absence of this binding between Lef/TCF transcription factors and -catenin, the Lef/TCF transcription factors are associated with members of the Groucho family of transcription repressor proteins (^{Akiyama, 2000}).

There is evidence to suggest that various proteins involved in the Wnt signaling pathway regulate epidermal stem cell fate decisions (^{DasGupta and Fuchs, 1999;Kishimoto et al, 2000}). Tcf3, a member of the Lef/TCF family, is expressed in cells in the bulge and outer root sheath regions of the hair follicle (^{Merrill et al, 2001}). As the Tcf3 positive cells in the bulge migrate along the hair follicle to the epidermis and sebaceous gland, Tcf3 expression is lost. This suggests that Tcf3 plays a role in epidermal stem cell maintenance.

Expression of another member of the Lef/TCF family, Lef1, is found in the hair producing progenitors of the hair follicle (^{DasGupta and Fuchs, 1999}). In transgenic mice that express stabilized -catenin in the epidermis there is apparent hair morphogenesis, which usually only takes place during embryogenesis when the dermal papilla and upper portion of the follicle are established (^{Gat et al, 1998}). Likewise, transgenic mice expressing Lef1 under control of the K14 promoter, which targets transgene expression to the hair follicle and basal layer of the epidermis, exhibited hair germ-like invaginations in the epidermis (^{Zhou et al, 1995}). In mice overexpressing a dominant-negative form of Lef1, a decrease in hair and an increase in sebaceous glands were observed. In addition, Lef1 knockout mice exhibit a marked reduction in the number of hair follicles (^{van Genderen et al, 1994}). Altogether, data from these mouse models indicate that -catenin/Lef1 transcriptional activation is required for hair differentiation.

c-Myc influences epidermal stem cell fate decisions

In addition to Tcf3 and Lef1, there is evidence that c-Myc plays a role in regulating epidermal stem cell fate decisions. c-Myc, a known target of Wnt signaling, is a proto-oncogene that encodes the Myc transcription factor known to induce growth, proliferation, transformation, and apoptosis (^{He et al, 1998;Eisenman, 2001}). The promoter of c-Myc contains binding sites for Tcf-4, which allow for induction of c-Myc expression by -catenin (^{He et al, 1998;Akiyama, 2000}). In the absence of -catenin, Tcf4 is able to bind to the c-Myc promoter and repress transcription (^{He et al, 1998}). It has been shown that c-Myc is able to induce differentiation of epidermal stem cells in vitro (^{Gandarillas and Watt, 1997}). Mice with targeted overexpression of c-Myc in the hair follicles and basal layer of the epidermis exhibit epidermal hyperplasia, hair loss, and an increase in sebaceous gland size and number (Figure 3) (^{Arnold and Watt, 2001;Waikel et al, 2001}). This suggests that c-Myc promotes the differentiation of stem cells into epidermal and sebaceous lineages at the expense of hair follicles.

Figure 3.

c-Myc transgenic mice exhibit an increase in the size and number of sebaceous glands. Oil Red O staining of sebaceous glands in normal adult mouse skin (left) and transgenic adult mouse skin (right) in which c-Myc is overexpressed in the basal layer of the epidermis and hair follicles. Note the increase in size and number of sebaceous glands in c-Myc transgenic mice. Modified from^{Waikel et al (2001)}.

Full figure and legend (42K)

c-Myc target genes

Although numerous putative target genes of c-Myc have been identified, the target genes of c-Myc that are involved in stem cell fate decisions are currently unknown. Besides its role in the regulation of stem cell fate decisions, c-Myc is involved in a wide variety of cellular processes, which is reflected by the identification of target genes involved in cell growth, proliferation, and apoptosis (^{Eisenman, 2001}). More specifically, the recent use of microarray technology has uncovered potential target genes of c-Myc that are required for cell cycle progression including genes involved in protein synthesis, lipid metabolism, DNA synthesis, protection from oxidative stress, and signal transduction (^{Schuhmacher et al, 2001}). In addition, microarray analysis of the expression profile of c-Myc-induced tumors identified genes involved in cell cycle progression as target genes of c-Myc in carcinogenesis (^{Schuldiner and Benvenisty, 2001}). Despite the identification of a vast array of c-Myc target genes, the target genes of c-Myc involved in stem cell fate decisions are still elusive.

Summary

As a downstream target of the Wnt signaling pathway, c-Myc is a potential candidate gene involved in stem cell fate decisions. Identification of target genes of c-Myc involved in epidermal and sebaceous gland differentiation will provide further insight into the mechanisms by which the Wnt signaling pathway controls stem cell fate decisions. In addition, understanding the role of p63 in epidermal development will help determine its impact on stem cell maintenance and epithelial differentiation. These studies, combined with others, will provide new insight into the regulatory mechanisms involved in epidermal development and differentiation and might identify therapeutic targets for a variety of skin disorders.

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Acknowledgements

This work was supported by National Institutes of Health grants AR62228, AR47898, CA52607, and HD25479 to D.R.R.