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Departments of Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, U.S.A.Departments of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, U.S.A.Departments of Dermatology, Baylor College of Medicine, Houston, Texas, U.S.A.
Epidermal development and differentiation are similar processes and therefore the study of one is likely to provide insight into the other. The signaling cascades required for epidermal differentiation are largely unknown. Recent evidence, however, has implicated two proteins, p63 and c-Myc, in different stages of epidermal development and differentiation. p63 was shown to be required for embryonic epidermal development. Mice lacking p63 do not develop stratified epithelia and appendages suggesting a role for p63 in the commitment to squamous epithelial lineages. Subsequent stem cell fate decisions are required to form the different structures of stratified epithelia including hair follicles, sebaceous glands, and epidermis. Several genes of the Wnt signaling pathway have been implicated in this process, including c-Myc, a downstream target of the Wnt pathway. Interestingly, targeted overexpression of c-Myc in the basal layer of the epidermis results in an increase in sebaceous gland size and number at the expense of hair follicles. This suggests that c-Myc promotes differentiation of epidermal stem cells into sebaceous glands. In this review, we discuss transgenic/knockout mouse models that have provided evidence linking c-Myc and p63 to different stages of epidermal development and differentiation.
The epidermis, which forms the outer layer of the skin, is a constantly self-renewing tissue that provides a fascinating system to study the molecular and cellular mechanisms governing tissue formation and homeostasis. Despite decades of research, relatively little is known about the regulatory pathways required for proper epidermal differentiation. These pathways regulate distinct processes such as epidermal stem cell fate decisions, initiation of epidermal differentiation, and transcriptional control of the differentiation process. Interestingly, the process of differentiation of normal adult epidermis shows striking similarities with the development of embryonic epidermis, reflected by the expression patterns of structural proteins in different compartments of the epidermis representing different stages of maturation. Based on these similarities, studies on skin development are likely to provide insight into the regulatory mechanisms governing skin differentiation. This review will focus on transgenic/knockout mouse models that have recently revealed the role of c-Myc and p63 in stem cell fate decisions and commitment to differentiation.
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 (
Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.
), 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). In addition, recent evidence suggests that p63 regulates the expression of several genes potentially involved in epidermal development and differentiation (
). 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) (
). 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 (ΔN isoforms). In addition, alternative splicing gives rise to three different carboxy termini designated α, β, and γ. The α carboxy terminus is the longest and contains a sterile α motif (SAM) domain (
). SAM domains are protein interaction motifs frequently found in proteins involved in development and differentiation, suggesting a role for p63α isoforms in these processes.
Figure 1Structure 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).
). 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 (
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 ΔN isoforms have a dominant-negative function (
). In addition, functional consequences of carboxy terminal variability have been described. Most notably, TAp63α, 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 (
. 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.
TAp63α was shown to induce expression of EphA2 tyrosine kinase, which inhibits integrin-mediated cell adhesion, potentially contributing to epidermal differentiation (
). 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 (
). 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 (
), 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 (
). The isoform targeted for overexpression appears to be ΔNp63α, 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 ΔNp63α under control of the mouse loricrin promoter suggesting that dominant-negative isoforms of p63 may play an oncogenic role in epithelial tissues (
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 (
). 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 ΔNp63γ isoform (
). 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 (
). 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 (
). Epidermal stem cells have been shown to express high levels of β1 integrin compared to transit amplifying cells. This has provided a way to isolate epidermal stem cells based on their adhesiveness to extracellular matrix proteins (
). 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 (
). 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
. 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 (
). 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 (
). 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 2Multipotent 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).
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 (
). 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 (
). 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-3β, thereby stabilizing β-catenin. In the absence of Wnt signaling, phosphorylation of β-catenin by glycogen synthase kinase-3β targets β-catenin for degradation via ubiquitination. β-Catenin, first identified as a cell-adhesion protein, is stabilized by Wnt signaling. After stabilization, β-catenin translocates to the nucleus where it binds to coactivators from the Lef/TCF family and activates the transcription of target genes (reviewed in
). In the absence of this binding between Lef/TCF transcription factors and β-catenin, the Lef/TCF transcription factors are associated with members of the Groucho family of transcription repressor proteins (
). 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 (
). In transgenic mice that express stabilized β-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 (
). 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 (
). 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 (
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 (
). 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) (
). This suggests that c-Myc promotes the differentiation of stem cells into epidermal and sebaceous lineages at the expense of hair follicles.
Figure 3c-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
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 (
). 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 (
). 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 (
). 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.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants AR62228, AR47898, CA52607, and HD25479 to D.R.R.
Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.
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