- Email: [email protected]
Knockout Mouse Models Reveal Genes Involved in Stem Cell Fate Decisions and Commitment to Differentiation
Epidermal Di¡erentiation: Transgenic/Knockout Mouse Models Reveal Genes Involved in Stem Cell Fate Decisions and Commitment to Di¡erentiation Maranke I. Koster,n Kimberly A. Huntzinger,w and Dennis R. Roopnwz Departments of wMolecular and Cellular Biology and zDermatology and nProgram in Developmental Biology, Baylor College of Medicine, Houston, Texas, U.S.A.
Epidermal development and di¡erentiation are similar processes and therefore the study of one is likely to provide insight into the other. The signaling cascades required for epidermal di¡erentiation are largely unknown. Recent evidence, however, has implicated two proteins, p63 and c-Myc, in di¡erent stages of epidermal development and di¡erentiation. p63 was shown to be required for embryonic epidermal development. Mice lacking p63 do not develop strati¢ed 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 di¡erent structures of strati¢ed 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 di¡erentiation 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 di¡erent stages of epidermal development and di¡erentiation. Key words: c-Myc/ epidermal di¡erentiation/p63/stem cells/transgenic/knockout mice. JID Symposium Proceedings 7:41 ^45, 2002
T
cross-talk between the ectoderm and the underlying mesenchyme. 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 strati¢cation, 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 ¢rst stage of epidermal di¡erentiation 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 ¢laggrin are ¢rst 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 strati¢ed and the periderm is shed.
he 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 di¡erentiation. These pathways regulate distinct processes such as epidermal stem cell fate decisions, initiation of epidermal di¡erentiation, and transcriptional control of the differentiation process. Interestingly, the process of di¡erentiation of normal adult epidermis shows striking similarities with the development of embryonic epidermis, re£ected by the expression patterns of structural proteins in di¡erent compartments of the epidermis representing di¡erent 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 di¡erentiation. EPIDERMAL DEVELOPMENT
p63 IN EPIDERMAL DEVELOPMENT
The signaling cascades responsible for the onset of epidermal development are poorly understood, but are likely to involve
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-strati¢ed epithelial structure required for limb bud outgrowth ( Johnson and Tabin, 1997; Mills et al, 1999; Yang et al, 1999). After E9.5, p63 continues
Manuscript accepted for publication July 12, 2002 Reprint requests to: Dennis R. Roop, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; Email: [email protected] Abbreviations: AER, apical ectodermal ridge; SAM, sterile a motif.
0022-202X/02/$15.00 Copyright r 2002 by The Society for Investigative Dermatology, Inc.
41
42
KOSTER ET AL
JID SYMPOSIUM PROCEEDINGS
to be expressed in the basal layer of the developing and adult epidermis and other strati¢ed and pseudo-strati¢ed 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 strati¢ed and pseudo-strati¢ed 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 di¡erentiation markers including K5, K14, K1, K10, loricrin, ¢laggrin, 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 strati¢ed 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 ectodermalmesenchymal 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 di¡erentiation markers loricrin, ¢laggrin, 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 di¡erentiation 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 strati¢ed 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, de¢nitive 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 di¡erentiated 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 di¡erentiation (Dohn et al, 2001; Nishi et al, 2001; Sasaki et al, 2001). Therefore, a plausible role for p63 is the induction of a squamous di¡erentiation program.
STRUCTURE OF p63
The understanding of the precise function of p63 in epithelial development is complicated by the ¢nding that p63 is transcribed into at least six di¡erent isoforms generated by alternative promoter usage and alternative splicing (Fig 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 (DN isoforms). In addition, alternative splicing gives rise to three di¡erent carboxy termini designated a, b, and g. The a carboxy terminus is the longest and contains a sterile a motif (SAM) domain (Chi et al, 1999). SAM domains are protein interaction motifs frequently found in proteins involved in development
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).
and di¡erentiation, suggesting a role for p63a isoforms in these processes.
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 di¡erent 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 a⁄nity for p53 response elements that contain more mismatched nucleotides (e.g., Bax-1), suggesting that p53 and p63 may have overlapping as well as di¡erent 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 DN isoforms have a dominant-negative function (Yang et al, 1998). In addition, functional consequences of carboxy terminal variability have been described. Most notably, TAp63a, 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 re£ect 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 identi¢ed target genes of p63 that are potentially involved in epidermal di¡erentiation and development. TAp63a was shown to induce expression of EphA2 tyrosine kinase, which inhibits integrin-mediated cell adhesion, potentially contributing to epidermal di¡erentiation (Miao et al, 2000; Dohn et al, 2001). In addition, TAp63g 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 di¡erentiation 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 di¡erentiation.
VOL. 7, NO. 1 DECEMBER 2002
EPIDERMAL DIFFERENTIATION
43
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 di¡erentiation 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 ampli¢cation 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 DNp63a, 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 DNp63a 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 a¡ect tissues of ectodermal origin. Mutations in p63 have been shown to underlie ectrodactyly, ectodermal dysplasia and cleft lip (EEC), limb^mammary syndrome (LMS), split handsplit foot malformation (SHFM), ankyloblepharon ectodermal dysplasia and clefting (AEC or HayWells disease), and acro-dermato-unguallacrimal^tooth syndrome (ADULT). Each of these syndromes has been shown to result from di¡erent 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 identi¢ed mutation were used for transactivation assays using a reporter gene under control of the p53 response element. Only subtle di¡erences 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 DNp63g 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 di¡erentiation program. A ¢rst 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 identi¢cation 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.
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 di¡erentiated 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 di¡erentiate giving rise to progeny at progressive stages of maturation (multiple shades of green).
and the epidermis ( Jones and Watt, 1993). Epidermal stem cells give rise to transit amplifying cells, which have a high potential to undergo di¡erentiation 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 b1 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 (Fig 2).
LOCATION OF EPIDERMAL STEM CELLS
Wnt SIGNALING REGULATES EPIDERMAL STEM CELL FATE DECISIONS
An equally important process required for epidermal development and di¡erentiation 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 di¡erentiation (Lajtha, 1979). Stem cells have been found in permanently renewing tissues including the hemopoietic system, the small intestine lining,
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
44
KOSTER ET AL
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-3b, thereby stabilizing b-catenin. In the absence of Wnt signaling, phosphorylation of b-catenin by glycogen synthase kinase-3b targets b-catenin for degradation via ubiquitination. b-Catenin, ¢rst identi¢ed as a cell-adhesion protein, is stabilized by Wnt signaling. After stabilization, b-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 b-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 b-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 b-catenin/Lef1 transcriptional activation is required for hair di¡erentiation. 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 b-catenin (He et al, 1998; Akiyama, 2000). In the absence of b-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
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 cMyc transgenic mice. Modi¢ed from Waikel et al (2001).
JID SYMPOSIUM PROCEEDINGS
di¡erentiation 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 (Fig 3) (Arnold and Watt, 2001 Waikel et al, 2001). This suggests that c-Myc promotes the di¡erentiation of stem cells into epidermal and sebaceous lineages at the expense of hair follicles. c-Myc TARGET GENES
Although numerous putative target genes of c-Myc have been identi¢ed, 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 re£ected by the identi¢cation of target genes involved in cell growth, proliferation, and apoptosis (Eisenman, 2001). More speci¢cally, 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 pro¢le of c-Myc-induced tumors identi¢ed genes involved in cell cycle progression as target genes of c-Myc in carcinogenesis (Schuldiner and Benvenisty, 2001). Despite the identi¢cation 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. Identi¢cation of target genes of c-Myc involved in epidermal and sebaceous gland di¡erentiation 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 di¡erentiation. These studies, combined with others, will provide new insight into the regulatory mechanisms involved in epidermal development and di¡erentiation and might identify therapeutic targets for a variety of skin disorders. This work was supported by National Institutes of Health grants AR62228, AR47898, CA52607, and HD25479 to D.R.R.
REFERENCES Akiyama T: Wnt/beta-catenin signaling. Cytokine Growth Factor Rev 11:273^282, 2000 Arnold I, Watt FM: c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and di¡erentiation of their progeny. Curr Biol 11:558^568, 2001 Barrandon Y, Green H: Three clonal types of keratinocyte with di¡erent capacities for multiplication. Proc Natl Acad Sci USA 84:2302^2306, 1987 Bian J, Sun Y: p53CP, a putative p53 competing protein that speci¢cally binds to the consensus p53 DNA binding sites: a third member of the p53 family? Proc Natl Acad Sci USA 94:14753^14758, 1997 Bickenbach JR, Chism E: Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res 244:184^195, 1998 Bickenbach JR, Greer JM, Bundman DS, Rothnagel JA, Roop DR: Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development. J Invest Dermatol 104:405^410, 1995 van Bokhoven H, Hamel BC, Bamshad M, et al: p63 gene mutations in eec syndrome, limb^mammary syndrome, and isolated split hand-split foot malformation suggest a genotypephenotype correlation. Am J Hum Genet 69:481^492, 2001 Byrne C, Tainsky M, Fuchs E: Programming gene expression in developing epidermis. Development 120:2369^2383, 1994
VOL. 7, NO. 1 DECEMBER 2002
Celli J, Duijf P, Hamel BC, et al: Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99:143^153, 1999 Chi SW, Ayed A, Arrowsmith CH: Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. EMBO J 18:4438^4445, 1999 Crook T, Nicholls JM, Brooks L, O’Nions J, Allday MJ: High level expression of deltaN-p63: a mechanism for the inactivation of p53 in undi¡erentiated nasopharyngeal carcinoma (NPC)? Oncogene 19:3439^3444, 2000 DasGupta R, Fuchs E: Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and di¡erentiation. Development 126:4557^ 4568, 1999 el Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B: De¢nition of a consensus binding site for p53. Nat Genet 1:45^49, 1992 Dohn M, Jiang J, Chen X: Receptor tyrosine kinase EphA2 is regulated by p53 -family proteins and induces apoptosis. Oncogene 20:6503^6515, 2001 Duijf PH, Vanmolkot KR, Propping P, et al: Gain-of-function mutation in ADULT syndrome reveals the presence of a second transactivation domain in p63. Hum Mol Genet 11:799^804, 2002 Eisenman RN: Deconstructing myc. Genes Dev 15:2023^2030, 2001 Friedrich G, Soriano P: Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513^1523, 1991 Gandarillas A, Watt FM: c-Myc promotes di¡erentiation of human epidermal stem cells. Genes Dev 11:2869^2882, 1997 Gat U, DasGupta R, Degenstein L, Fuchs E: De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95:605^614, 1998 van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, Grosschedl R: Development of several organs that require inductive epithelial^mesenchymal interactions is impaired in LEF-1-de¢cient mice. Genes Dev 8:2691^2703, 1994 Hagiwara K, McMenamin MG, Miura K, Harris CC: Mutational analysis of the p63/ p73L/p51/p40/CUSP/KET gene in human cancer cell lines using intronic primers. Cancer Res 59:4165^4169, 1999 Hardy MH: The secret life of the hair follicle. Trends Genet 8:55^61, 1992 He TC, Sparks AB, Rago C, et al: Identi¢cation of c-Myc as a target of the APC pathway. Science 281:1509^1512, 1998 Hibi K, Trink B, Patturajan M, et al: AIS is an oncogene ampli¢ed in squamous cell carcinoma. Proc Natl Acad Sci USA 97:5462^5467, 2000 Ianakiev P, Kilpatrick MW, Toudjarska I, Basel D, Beighton P, Tsipouras P: Splithand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am J Hum Genet 67:59^66, 2000 Jackson BW, Grund C, Winter S, Franke WW, Illmensee K: Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial di¡erentiation and intermediate-sized ¢laments in early postimplantation embryos. Di¡erentiation 20:203^216, 1981 Johnson RL, Tabin CJ: Molecular models for vertebrate limb development. Cell 90:979^990, 1997 Jones PH, Watt FM: Separation of human epidermal stem cells from transit amplifying cells on the basis of di¡erences in integrin function and expression. Cell 73:713^724, 1993 Kishimoto J, Burgeson RE, Morgan BA: Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 14:1181^1185, 2000 Lajtha LG: Stem cell concepts. Di¡erentiation 14:23^34, 1979 Liefer KM, Koster MI, Wang XJ, Yang A, McKeon F, Roop DR: Down-regulation of p63 is required for epidermal UV-B-induced apoptosis. Cancer Res 60:4016^ 4020, 2000 Lowell S, Jones P, Le RI, Dunne J,Watt FM: Stimulation of human epidermal di¡erentiation by delta-notch signalling at the boundaries of stem-cell clusters. Curr Biol 10:491^500, 2000 M’Boneko V, Merker HJ: Development and morphology of the periderm of mouse embryos (days 9^12 of gestation). Acta Anat (Basel) 133:325^336, 1988
EPIDERMAL DIFFERENTIATION
45
McGrath JA, Duijf PH, Doetsch V, et al: Hay^Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum Mol Genet 10: 221^229, 2001 Mehrel T, Hohl D, Rothnagel JA, et al: Identi¢cation of a major keratinocyte cell envelope protein, loricrin. Cell 61:1103^1112, 1990 Merrill BJ, Gat U, DasGupta R, Fuchs E: Tcf3 and Lef1 regulate lineage di¡erentiation of multipotent stem cells in skin. Genes Dev 15:1688^1705, 2001 Miao H, Burnett E, Kinch M, Simon E, Wang B: Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol 2:62^69, 2000 Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398:708^713, 1999 Nishi H, Senoo M, Nishi KH, et al: p53 homologue p63 represses epidermal growth factor receptor expression. J Biol Chem 276:41717^41724, 2001 Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y: Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233^245, 2001 Park BJ, Lee SJ, Kim JI, et al: Frequent alteration of p63 expression in human primary bladder carcinomas. Cancer Res 60:3370^3374, 2000 Pellegrini G, Dellambra E, Golisano O, et al: p63 identi¢es keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156^3161, 2001 Polakis P: Wnt signaling and cancer. Genes Dev 14:1837^1851, 2000 Powell BC, Passmore EA, Nesci A, Dunn SM: The Notch signalling pathway in hair growth. Mech Dev 78:189^192, 1998 Reynolds AJ, Jahoda CA: Hair follicle stem cells? A distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells. J Cell Sci 99:373^385, 1991 Rothnagel JA, Mehrel T, Idler WW, Roop DR, Steinert PM: The gene for mouse epidermal ¢laggrin precursor. Its partial characterization, expression, and sequence of a repeating ¢laggrin unit. J Biol Chem 262:15643^15648, 1987 Sasaki Y, Ishida S, Morimoto I, et al: The p53 family member genes are involved in the Notch signal pathway. J Biol Chem 277:719^724, 2002 Schuhmacher M, Kohlhuber F, Holzel M, et al: The transcriptional program of a human B cell line in response to Myc. Nucl Acids Res 29:397^406, 2001 Schuldiner O, Benvenisty N: A DNA microarray screen for genes involved in c-Myc and N-Myc oncogenesis in human tumors. Oncogene 20:4984^4994, 2001 Smith CL, Hager GL: Transcriptional regulation of mammalian genes in vivo . A tale of two templates. J Biol Chem 272:27493^27496, 1997 Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM: Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451^461, 2000 Waikel RL, Kawachi Y, Waikel PA, Wang XJ, Roop DR: Deregulated expression of c-Myc depletes epidermal stem cells. Nat Genet 28:165^168, 2001 Yamaguchi K, Wu L, Caballero OL, et al: Frequent gain of the p40/p51/p63 gene locus in primary head and neck squamous cell carcinoma. Int J Cancer 86:684^ 689, 2000 Yang A, Kaghad M, Wang Y, et al: p63, a p53 homolog at 3q27^29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2:305^316, 1998 Yang A, Schweitzer R, Sun D, et al: p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398:714^718, 1999 Zambruno G, Girolomoni G, Manca V, Segre A, Giannetti A: Epidermal growth factor and transferrin receptor expression in human embryonic and fetal epidermal cells. Arch Dermatol Res 282:544^548, 1990 Zeng X, Levine AJ, Lu H: Non-p53 p53RE binding protein, a human transcription factor functionally analogous to p53. Proc Natl Acad Sci USA 95:6681^6686, 1998 Zhou P, Byrne C, Jacobs J, Fuchs E: Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev 9:700^713, 1995
Epidermal development and di¡erentiation are similar processes and therefore the study of one is likely to provide insight into the other. The signaling cascades required for epidermal di¡erentiation are largely unknown. Recent evidence, however, has implicated two proteins, p63 and c-Myc, in di¡erent stages of epidermal development and di¡erentiation. p63 was shown to be required for embryonic epidermal development. Mice lacking p63 do not develop strati¢ed 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 di¡erent structures of strati¢ed 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 di¡erentiation 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 di¡erent stages of epidermal development and di¡erentiation. Key words: c-Myc/ epidermal di¡erentiation/p63/stem cells/transgenic/knockout mice. JID Symposium Proceedings 7:41 ^45, 2002
T
cross-talk between the ectoderm and the underlying mesenchyme. 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 strati¢cation, 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 ¢rst stage of epidermal di¡erentiation 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 ¢laggrin are ¢rst 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 strati¢ed and the periderm is shed.
he 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 di¡erentiation. These pathways regulate distinct processes such as epidermal stem cell fate decisions, initiation of epidermal di¡erentiation, and transcriptional control of the differentiation process. Interestingly, the process of di¡erentiation of normal adult epidermis shows striking similarities with the development of embryonic epidermis, re£ected by the expression patterns of structural proteins in di¡erent compartments of the epidermis representing di¡erent 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 di¡erentiation. EPIDERMAL DEVELOPMENT
p63 IN EPIDERMAL DEVELOPMENT
The signaling cascades responsible for the onset of epidermal development are poorly understood, but are likely to involve
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-strati¢ed epithelial structure required for limb bud outgrowth ( Johnson and Tabin, 1997; Mills et al, 1999; Yang et al, 1999). After E9.5, p63 continues
Manuscript accepted for publication July 12, 2002 Reprint requests to: Dennis R. Roop, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; Email: [email protected] Abbreviations: AER, apical ectodermal ridge; SAM, sterile a motif.
0022-202X/02/$15.00 Copyright r 2002 by The Society for Investigative Dermatology, Inc.
41
42
KOSTER ET AL
JID SYMPOSIUM PROCEEDINGS
to be expressed in the basal layer of the developing and adult epidermis and other strati¢ed and pseudo-strati¢ed 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 strati¢ed and pseudo-strati¢ed 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 di¡erentiation markers including K5, K14, K1, K10, loricrin, ¢laggrin, 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 strati¢ed 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 ectodermalmesenchymal 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 di¡erentiation markers loricrin, ¢laggrin, 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 di¡erentiation 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 strati¢ed 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, de¢nitive 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 di¡erentiated 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 di¡erentiation (Dohn et al, 2001; Nishi et al, 2001; Sasaki et al, 2001). Therefore, a plausible role for p63 is the induction of a squamous di¡erentiation program.
STRUCTURE OF p63
The understanding of the precise function of p63 in epithelial development is complicated by the ¢nding that p63 is transcribed into at least six di¡erent isoforms generated by alternative promoter usage and alternative splicing (Fig 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 (DN isoforms). In addition, alternative splicing gives rise to three di¡erent carboxy termini designated a, b, and g. The a carboxy terminus is the longest and contains a sterile a motif (SAM) domain (Chi et al, 1999). SAM domains are protein interaction motifs frequently found in proteins involved in development
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).
and di¡erentiation, suggesting a role for p63a isoforms in these processes.
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 di¡erent 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 a⁄nity for p53 response elements that contain more mismatched nucleotides (e.g., Bax-1), suggesting that p53 and p63 may have overlapping as well as di¡erent 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 DN isoforms have a dominant-negative function (Yang et al, 1998). In addition, functional consequences of carboxy terminal variability have been described. Most notably, TAp63a, 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 re£ect 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 identi¢ed target genes of p63 that are potentially involved in epidermal di¡erentiation and development. TAp63a was shown to induce expression of EphA2 tyrosine kinase, which inhibits integrin-mediated cell adhesion, potentially contributing to epidermal di¡erentiation (Miao et al, 2000; Dohn et al, 2001). In addition, TAp63g 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 di¡erentiation 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 di¡erentiation.
VOL. 7, NO. 1 DECEMBER 2002
EPIDERMAL DIFFERENTIATION
43
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 di¡erentiation 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 ampli¢cation 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 DNp63a, 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 DNp63a 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 a¡ect tissues of ectodermal origin. Mutations in p63 have been shown to underlie ectrodactyly, ectodermal dysplasia and cleft lip (EEC), limb^mammary syndrome (LMS), split handsplit foot malformation (SHFM), ankyloblepharon ectodermal dysplasia and clefting (AEC or HayWells disease), and acro-dermato-unguallacrimal^tooth syndrome (ADULT). Each of these syndromes has been shown to result from di¡erent 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 identi¢ed mutation were used for transactivation assays using a reporter gene under control of the p53 response element. Only subtle di¡erences 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 DNp63g 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 di¡erentiation program. A ¢rst 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 identi¢cation 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.
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 di¡erentiated 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 di¡erentiate giving rise to progeny at progressive stages of maturation (multiple shades of green).
and the epidermis ( Jones and Watt, 1993). Epidermal stem cells give rise to transit amplifying cells, which have a high potential to undergo di¡erentiation 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 b1 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 (Fig 2).
LOCATION OF EPIDERMAL STEM CELLS
Wnt SIGNALING REGULATES EPIDERMAL STEM CELL FATE DECISIONS
An equally important process required for epidermal development and di¡erentiation 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 di¡erentiation (Lajtha, 1979). Stem cells have been found in permanently renewing tissues including the hemopoietic system, the small intestine lining,
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
44
KOSTER ET AL
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-3b, thereby stabilizing b-catenin. In the absence of Wnt signaling, phosphorylation of b-catenin by glycogen synthase kinase-3b targets b-catenin for degradation via ubiquitination. b-Catenin, ¢rst identi¢ed as a cell-adhesion protein, is stabilized by Wnt signaling. After stabilization, b-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 b-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 b-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 b-catenin/Lef1 transcriptional activation is required for hair di¡erentiation. 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 b-catenin (He et al, 1998; Akiyama, 2000). In the absence of b-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
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 cMyc transgenic mice. Modi¢ed from Waikel et al (2001).
JID SYMPOSIUM PROCEEDINGS
di¡erentiation 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 (Fig 3) (Arnold and Watt, 2001 Waikel et al, 2001). This suggests that c-Myc promotes the di¡erentiation of stem cells into epidermal and sebaceous lineages at the expense of hair follicles. c-Myc TARGET GENES
Although numerous putative target genes of c-Myc have been identi¢ed, 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 re£ected by the identi¢cation of target genes involved in cell growth, proliferation, and apoptosis (Eisenman, 2001). More speci¢cally, 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 pro¢le of c-Myc-induced tumors identi¢ed genes involved in cell cycle progression as target genes of c-Myc in carcinogenesis (Schuldiner and Benvenisty, 2001). Despite the identi¢cation 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. Identi¢cation of target genes of c-Myc involved in epidermal and sebaceous gland di¡erentiation 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 di¡erentiation. These studies, combined with others, will provide new insight into the regulatory mechanisms involved in epidermal development and di¡erentiation and might identify therapeutic targets for a variety of skin disorders. This work was supported by National Institutes of Health grants AR62228, AR47898, CA52607, and HD25479 to D.R.R.
REFERENCES Akiyama T: Wnt/beta-catenin signaling. Cytokine Growth Factor Rev 11:273^282, 2000 Arnold I, Watt FM: c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and di¡erentiation of their progeny. Curr Biol 11:558^568, 2001 Barrandon Y, Green H: Three clonal types of keratinocyte with di¡erent capacities for multiplication. Proc Natl Acad Sci USA 84:2302^2306, 1987 Bian J, Sun Y: p53CP, a putative p53 competing protein that speci¢cally binds to the consensus p53 DNA binding sites: a third member of the p53 family? Proc Natl Acad Sci USA 94:14753^14758, 1997 Bickenbach JR, Chism E: Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res 244:184^195, 1998 Bickenbach JR, Greer JM, Bundman DS, Rothnagel JA, Roop DR: Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development. J Invest Dermatol 104:405^410, 1995 van Bokhoven H, Hamel BC, Bamshad M, et al: p63 gene mutations in eec syndrome, limb^mammary syndrome, and isolated split hand-split foot malformation suggest a genotypephenotype correlation. Am J Hum Genet 69:481^492, 2001 Byrne C, Tainsky M, Fuchs E: Programming gene expression in developing epidermis. Development 120:2369^2383, 1994
VOL. 7, NO. 1 DECEMBER 2002
Celli J, Duijf P, Hamel BC, et al: Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99:143^153, 1999 Chi SW, Ayed A, Arrowsmith CH: Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. EMBO J 18:4438^4445, 1999 Crook T, Nicholls JM, Brooks L, O’Nions J, Allday MJ: High level expression of deltaN-p63: a mechanism for the inactivation of p53 in undi¡erentiated nasopharyngeal carcinoma (NPC)? Oncogene 19:3439^3444, 2000 DasGupta R, Fuchs E: Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and di¡erentiation. Development 126:4557^ 4568, 1999 el Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B: De¢nition of a consensus binding site for p53. Nat Genet 1:45^49, 1992 Dohn M, Jiang J, Chen X: Receptor tyrosine kinase EphA2 is regulated by p53 -family proteins and induces apoptosis. Oncogene 20:6503^6515, 2001 Duijf PH, Vanmolkot KR, Propping P, et al: Gain-of-function mutation in ADULT syndrome reveals the presence of a second transactivation domain in p63. Hum Mol Genet 11:799^804, 2002 Eisenman RN: Deconstructing myc. Genes Dev 15:2023^2030, 2001 Friedrich G, Soriano P: Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513^1523, 1991 Gandarillas A, Watt FM: c-Myc promotes di¡erentiation of human epidermal stem cells. Genes Dev 11:2869^2882, 1997 Gat U, DasGupta R, Degenstein L, Fuchs E: De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95:605^614, 1998 van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, Grosschedl R: Development of several organs that require inductive epithelial^mesenchymal interactions is impaired in LEF-1-de¢cient mice. Genes Dev 8:2691^2703, 1994 Hagiwara K, McMenamin MG, Miura K, Harris CC: Mutational analysis of the p63/ p73L/p51/p40/CUSP/KET gene in human cancer cell lines using intronic primers. Cancer Res 59:4165^4169, 1999 Hardy MH: The secret life of the hair follicle. Trends Genet 8:55^61, 1992 He TC, Sparks AB, Rago C, et al: Identi¢cation of c-Myc as a target of the APC pathway. Science 281:1509^1512, 1998 Hibi K, Trink B, Patturajan M, et al: AIS is an oncogene ampli¢ed in squamous cell carcinoma. Proc Natl Acad Sci USA 97:5462^5467, 2000 Ianakiev P, Kilpatrick MW, Toudjarska I, Basel D, Beighton P, Tsipouras P: Splithand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am J Hum Genet 67:59^66, 2000 Jackson BW, Grund C, Winter S, Franke WW, Illmensee K: Formation of cytoskeletal elements during mouse embryogenesis. II. Epithelial di¡erentiation and intermediate-sized ¢laments in early postimplantation embryos. Di¡erentiation 20:203^216, 1981 Johnson RL, Tabin CJ: Molecular models for vertebrate limb development. Cell 90:979^990, 1997 Jones PH, Watt FM: Separation of human epidermal stem cells from transit amplifying cells on the basis of di¡erences in integrin function and expression. Cell 73:713^724, 1993 Kishimoto J, Burgeson RE, Morgan BA: Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 14:1181^1185, 2000 Lajtha LG: Stem cell concepts. Di¡erentiation 14:23^34, 1979 Liefer KM, Koster MI, Wang XJ, Yang A, McKeon F, Roop DR: Down-regulation of p63 is required for epidermal UV-B-induced apoptosis. Cancer Res 60:4016^ 4020, 2000 Lowell S, Jones P, Le RI, Dunne J,Watt FM: Stimulation of human epidermal di¡erentiation by delta-notch signalling at the boundaries of stem-cell clusters. Curr Biol 10:491^500, 2000 M’Boneko V, Merker HJ: Development and morphology of the periderm of mouse embryos (days 9^12 of gestation). Acta Anat (Basel) 133:325^336, 1988
EPIDERMAL DIFFERENTIATION
45
McGrath JA, Duijf PH, Doetsch V, et al: Hay^Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum Mol Genet 10: 221^229, 2001 Mehrel T, Hohl D, Rothnagel JA, et al: Identi¢cation of a major keratinocyte cell envelope protein, loricrin. Cell 61:1103^1112, 1990 Merrill BJ, Gat U, DasGupta R, Fuchs E: Tcf3 and Lef1 regulate lineage di¡erentiation of multipotent stem cells in skin. Genes Dev 15:1688^1705, 2001 Miao H, Burnett E, Kinch M, Simon E, Wang B: Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol 2:62^69, 2000 Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398:708^713, 1999 Nishi H, Senoo M, Nishi KH, et al: p53 homologue p63 represses epidermal growth factor receptor expression. J Biol Chem 276:41717^41724, 2001 Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y: Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233^245, 2001 Park BJ, Lee SJ, Kim JI, et al: Frequent alteration of p63 expression in human primary bladder carcinomas. Cancer Res 60:3370^3374, 2000 Pellegrini G, Dellambra E, Golisano O, et al: p63 identi¢es keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156^3161, 2001 Polakis P: Wnt signaling and cancer. Genes Dev 14:1837^1851, 2000 Powell BC, Passmore EA, Nesci A, Dunn SM: The Notch signalling pathway in hair growth. Mech Dev 78:189^192, 1998 Reynolds AJ, Jahoda CA: Hair follicle stem cells? A distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells. J Cell Sci 99:373^385, 1991 Rothnagel JA, Mehrel T, Idler WW, Roop DR, Steinert PM: The gene for mouse epidermal ¢laggrin precursor. Its partial characterization, expression, and sequence of a repeating ¢laggrin unit. J Biol Chem 262:15643^15648, 1987 Sasaki Y, Ishida S, Morimoto I, et al: The p53 family member genes are involved in the Notch signal pathway. J Biol Chem 277:719^724, 2002 Schuhmacher M, Kohlhuber F, Holzel M, et al: The transcriptional program of a human B cell line in response to Myc. Nucl Acids Res 29:397^406, 2001 Schuldiner O, Benvenisty N: A DNA microarray screen for genes involved in c-Myc and N-Myc oncogenesis in human tumors. Oncogene 20:4984^4994, 2001 Smith CL, Hager GL: Transcriptional regulation of mammalian genes in vivo . A tale of two templates. J Biol Chem 272:27493^27496, 1997 Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM: Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451^461, 2000 Waikel RL, Kawachi Y, Waikel PA, Wang XJ, Roop DR: Deregulated expression of c-Myc depletes epidermal stem cells. Nat Genet 28:165^168, 2001 Yamaguchi K, Wu L, Caballero OL, et al: Frequent gain of the p40/p51/p63 gene locus in primary head and neck squamous cell carcinoma. Int J Cancer 86:684^ 689, 2000 Yang A, Kaghad M, Wang Y, et al: p63, a p53 homolog at 3q27^29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2:305^316, 1998 Yang A, Schweitzer R, Sun D, et al: p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398:714^718, 1999 Zambruno G, Girolomoni G, Manca V, Segre A, Giannetti A: Epidermal growth factor and transferrin receptor expression in human embryonic and fetal epidermal cells. Arch Dermatol Res 282:544^548, 1990 Zeng X, Levine AJ, Lu H: Non-p53 p53RE binding protein, a human transcription factor functionally analogous to p53. Proc Natl Acad Sci USA 95:6681^6686, 1998 Zhou P, Byrne C, Jacobs J, Fuchs E: Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev 9:700^713, 1995