review trends in cell biology vol.12 no.4 april 2002...

TRENDS in Cell Biology Vol.12 No.4 April 2002

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185Review

Catherin Niemann

Fiona M. Watt*

Keratinocyte Laboratory,Cancer Research UK, 44 Lincoln’s Inn Fields,London, UK WC2A 3PX.*e-mail: [email protected]

The epidermis forms the protective outer covering ofmammalian skin. It comprises a multilayeredepithelium, the interfollicular epidermis (IFE),associated hair follicles and sebaceous and sweatglands (Fig. 1). The epidermis is maintainedthroughout adult life by stem cells that not onlyself-renew but also generate daughter cells thatundergo terminal differentiation (a process ofdifferentiation resulting in a highly specialized cell that cannot divide). There is a single terminaldifferentiation pathway within the IFE, albeit one that varies slightly between sites in the body. This culminates in the production of the outermost,cornified epidermal layers that provide a protectivecovering for the skin. The terminally differentiatedcells of the sebaceous gland are lipid-filled sebocytesthat eventually burst, releasing their contents onto the surface of the skin. The hair follicle is more complex, comprising eight different cell lineages (Fig. 2).

The availability of promoters to target transgenesto specific compartments of mouse epidermis or to generate epidermal-specific knockouts has resultedin a wealth of data about the factors that regulateepidermal growth, differentiation and homeostasis[1,2]. Our purpose here is to highlight one aspect ofepidermal differentiation, namely the regulation oflineage commitment (the decision to undergo aspecific programme of terminal differentiation) inpostnatal life. Put simply, an understanding oflineage commitment provides the prospect ofengineering the epidermis to produce more hairfollicles for balding men, to banish excess sebumproduction for teenagers and to ensure smooth, hair-free legs for women. More importantly, it is becoming clear that the diversity of epidermaltumour types reflects the activity of molecules thatregulate lineage commitment and this knowledgemight be exploited to design tumour-specific anti-cancer drugs.

Epidermal stem cells, transit amplifying cells and

committed progenitors

It is well known that epidermal stem cells aremultipotent [3,4]. Stem cells in the hair follicle cangive rise not only to the hair lineages but also tosebocytes and IFE [5,6]. Conversely, with appropriatemesenchymal stimuli, cells of the IFE can be inducedto differentiate into hair and sebocyte lineages [7,8].Less is known about the origin of the cells of the sweat glands, although they too are probably formedby the differentiated progeny of multipotentepidermal stem cells and, under some conditions,cells from sweat glands can reconstitute a stratifiedsquamous epithelium [9].

It has been proposed that the stem cells that lie ina specialized region of the hair follicle outer rootsheath (ORS), known as the bulge, are ultimatelyresponsible for replenishing the differentiated cells of the IFE and sebaceous gland in addition togenerating all the hair lineages [5,6]. The bulge liesbeneath the sebaceous glands at the point of insertionof the arrector pili muscle (Fig. 1). However, recentlong-term lineage marking has demonstrated that, inundamaged epidermis, there are distinct stem-cellpopulations in the IFE, sebaceous gland and hair [10].Thus, although bulge stem cells have the ability togenerate all the different epidermal lineages, it islikely that, under steady-state conditions, stem cells

The epidermis is populated by stem cells that produce daughters that

differentiate to form the interfollicular epidermis, hair follicles and sebaceous

glands. Diffusible factors, cell–cell contact and extracellular matrix proteins are

all important components of the microenvironment of individual stem cells and

profoundly affect the differentiation pathways selected by their progeny. Here,

we summarize what is known about stem-cell populations and lineage

relationships within the epidermis. We also present evidence that postnatal

epidermis can be reprogrammed, altering the number and location of cells that

differentiate along specific epidermal lineages.

Designer skin: lineage commitment in

postnatal epidermis

Catherin Niemann and Fiona M. Watt

IFE

SGB

HF

Fig. 1. Haematoxylin- and eosin-stained section through the dorsal skinof an adult mouse during the anagen (growth) phase of the hair folliclecycle. Skin at this body site lacks sweat glands. Abbreviations: B,approximate location of the bulge; HF, hair follicle; IFE, interfollicularepidermis; SG, sebaceous gland. Bar, 200 µm.

feed only one or a subset of lineages, the choice oflineage being determined by the specific location, ormicroenvironment, of the cells.

Our view of the stem cell compartment isillustrated in Fig. 3. Separate stem cells for IFE, hair lineages and sebaceous gland are shown as beinginterconvertible, to emphasize that each populationhas multipotent differentiation capacity. Constituents of the microenvironment that coulddictate the differentiation pathways selected bystem-cell progeny include the extracellular matrix,diffusible factors (growth factors, cytokines andmorphogens), direct contact with neighbouring cells,the availability of oxygen and nutrients, and evenmechanical stimuli such as stretch forces [11,12]. It has long been appreciated that communicationwith the underlying dermis plays an important role in regulating epidermal differentiation [13], withthe specialized mesenchymal cells of the dermalpapilla at the base of the hair follicle (Fig. 2) providingthe best-characterized microenvironment (oftenreferred to as a niche) both in vivo [1,4] and in vitro[7,14]. Growth and differentiation of postnatal hairfollicles are controlled by reciprocal interactionsbetween the dermal papilla and the cells of the hair matrix (Fig. 2).

How are epidermal stem cells identified? Inundamaged adult epidermis, the stem cells divide

infrequently, although they have a high capacity for self-renewal [15]. In the mouse, they can thereforebe identified by injecting neonatal animals repeatedlywith 3H-thymidine or bromodeoxyuridine (BrdU) tolabel all the epidermal cells at a time when the tissueis maximally proliferative and then finding the cellsthat retain label in adulthood. In human IFE, thestem cells are found among those basal cells that areneither actively cycling nor committed to differentiate[16,17]. Although stem cells divide infrequently inadult epidermis, they can found actively proliferatingclones in culture and, in rodents, there is a goodcorrelation between the populations of keratinocytesidentified as stem cells by label retention and byclonal analysis [6,18]. In addition, several cell surface markers can be used to enrich for clonogenicor label-retaining keratinocytes directly from theepidermis [18].

If stem cells are cells that divide infrequently, what are the actively dividing cells withinmammalian epidermis? The most widely acceptedmodel is that, in the IFE, they are the non-stemdaughters of stem cells and that they divide a fewtimes before the onset of terminal differentiation.Such dividing cells are known as transit [19], ortransient [20], amplifying cells (Fig. 3). As the nameimplies, their role is to increase the number ofterminally differentiated cells produced by eachstem-cell division. Thus, even though stem cells havea high self-renewal capacity, they actually divideinfrequently in normal undamaged epidermis.

The significance of the transit amplifyingpopulation in terms of lineage commitment is lessclear. By analogy with haematopoiesis [21], eachtransit amplifying cell might be committed todifferentiate along only one or a few lineages; that is,transit amplifying cells might be ‘committedprogenitors’ (Fig. 3, brown). Alternatively, transitamplifying cells might be multipotent and differ fromstem cells only in their self-renewal ability. There isno fixed relationship between the timing ofirreversible exit from the cell cycle and the onset ofepidermal terminal differentiation [22]. Therefore,within a given lineage, there might be more than one class of transit amplifying cell, differing from oneanother in their expression of early differentiationmarkers, as proposed recently for human oesophagealepithelium [23]. Although it is convenient to think of stem and transit amplifying cells as discretepopulations, there might instead be gradients of cell behaviour [24], ranging from a cell that has maximum self-renewal capacity and zerodifferentiation probability (Fig. 3, red) to one that hascompleted terminal differentiation and can neverdivide again (Fig. 3, green); transit amplifying cellswould then lie somewhere in the middle of eachgradient [25].

The epidermis has not been ignored in the recentflurry of interest in adult stem cell plasticity. It hasnow been shown that haematopoietic stem cells can


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186 Review

TRENDS in Cell Biology

IRS

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Fig. 2. Hair follicle lineages. (a) Haematoxylin- and eosin-stained section through the base of agrowing follicle. (b) The different hair follicle lineages; the dermal papilla is shown in dark grey andthe hair matrix in light grey. Abbreviations: IRS, inner root sheath; ORS, outer root sheath. Bar, 50 µm.

give rise to cells within the hair follicle and IFE [26].Conversely, stem cells from neonatal mouseepidermis injected into mouse blastocysts contributeto ectoderm-, mesenchyme- and neural-crest-derivedtissues in adulthood, including neurons,haematopoietic cells and endothelium [27]. The transit amplifying compartment in adult

rabbit and mouse corneal epithelium can bereprogrammed by exposure to embryonic mousedermis to generate hair, sebocytes, sweat glands and IFE, showing not only differentiation plasticitybut also, potentially, differentiation reversal tomultipotent stem cells [28]. By contrast, epidermaltransit amplifying cells cannot contribute to any tissues after mouse blastocyst injection [27].Although these observations reveal that epidermalstem and transit amplifying cells can bereprogrammed under some circumstances, they do not imply that reprogramming occurs undernormal conditions. Environmental cues are ofparamount importance in regulating cell fate [11,12] and, because keratinocytes do not normallyleave the epidermis, it would be surprising ifdifferentiation into non-epithelial cell types occurswith any measurable frequency.

Stem-cell locations and lineage relationships

Where does lineage commitment take place? For theIFE, this question is relatively easy to answerbecause cells that have begun to express terminaldifferentiation markers can be readily identified in the epidermal basal layer. In human epidermis ofnon-hair-bearing skin, basal cells that are not activelycycling and express several putative stem-cellmarkers are found in clusters that have a specifictopology with respect to the underlying connectivetissue [16,17,29]. The stem-cell clusters aresurrounded by basal cells that are actively cycling and express markers of transit amplifying cells, and itis from within this latter compartment that cellscommitted to terminal differentiation move upwardsinto the layers above.

The location of lineage-committed progenitorswithin the hair follicle is harder to determine than for the IFE, owing partly to the existence of multiplehair lineages, partly to the fact that hair folliclegrowth is cyclical rather than continuous and partly to controversy surrounding the number of stem-cell populations within the hair follicle. One view is that there is a single location of hairfollicle stem cells, namely the bulge (Fig. 3a). At the end of each hair growth cycle, the specializedmesenchymal cells of the dermal papilla at the base of each follicle (Fig. 2a) come into close proximity with the bulge, triggering the bulge stem cells to divide and generate progenitors of all the differentiated cells in the growing follicle (the bulge activation hypothesis [1,30]). Alternatively,stem cells themselves might exit the bulge and migrate along the ORS to the base of thedeveloping follicle (the stem-cell migration hypothesis [1,6]).

A different view is that there are two stem-cellpopulations in the hair follicle, one in the bulge and one at the base of the follicle in a region known as the hair matrix or hair germ (the hair-folliclepredetermination model [4]) (Fig. 3b). The hair shaft



187Review

HSprogenitors

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HSprogenitors

IRSprogenitors

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SebocyteHenleHuxley

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nMulti-potential stem cell compartmentModel 1(a)

Multi-potential stem cell compartmentModel 2(b)

IFEstemcell

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Medulla ORSCortex

Fig. 3. Models of lineage relationships within the epidermis. Red denotes stem cells, green denotescells that have completed their programme of terminal differentiation and brown denotesintermediate populations (transit amplifying or progenitor cells). Curved arrows indicate unlimited orextensive self-renewal ability and double-headed arrows reflect the ability of any given stem cell toproduce daughter cells that differentiate along any lineage. Thus, although we envisage discretestem-cell populations, they together compose a multipotent stem-cell compartment (boxed). (a) InModel 1, there is only one hair-follicle stem-cell compartment, the bulge [5,6]. (b) In Model 2, there aretwo hair-follicle stem-cell compartments, one in the bulge and one in the matrix. The single-headedarrow connecting the follicle stem cells in (b) denotes possible replenishment of matrix stem cellsfrom the bulge [4], and the broken arrow reflects uncertainty about whether matrix cells replenish thebulge under some circumstances. Both models show what we believe to be the most likely origin ofthe companion layer [37]. Abbreviations: CL, companion layer; HF, hair follicle; HS, hair shaft; IFE,interfollicular epidermis; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland.

and inner root sheath (IRS) lineages would be derivedfrom the matrix stem-cell population, whereas thebulge stem cells would give rise to the ORS (Fig. 3b).This model proposes that, once the downward growthphase of the hair follicle is complete, stem cells fromthe bulge move down the ORS to form a distinctpopulation of cells at the periphery of the hair bulb.These cells survive the apoptosis that leads to loss ofthe lower part of the hair follicle at the end of hairgrowth and form a reservoir of stem cells that respondto signals from the dermal papilla to replenish theIRS and hair shaft when the new hair cycle begins. In support of this model, lineage marking studieshave established the existence of long-lived cells with multilineage differentiation capacity in the hair matrix [10,31].

Irrespective of whether there are one or tworeservoirs of stem cells in the hair follicle, clusteringof stem cells at specific sites of the IFE and follicleimplies lateral migration of their progeny. There isnow good evidence for this both within the hair follicle [6,31] and in human epidermis reconstitutedin vitro [18]. In both situations, there is an inverserelationship between stem-cell motility and in vitroclonogenicity [6,17]. Some of the proposed markers of the human epidermal stem cell compartment, suchas β1 integrins and the Notch ligand Delta1, mightpromote stem-cell clustering by restricting motility or stimulating intercellular cohesiveness and thus act as dual regulators of cell adhesion anddifferentiation [17,32,33].

Commitment to the three hair-shaft lineages(medulla, cortex, cuticle) and three IRS lineages (cuticle, Huxley’s layer and Henle’s layer)takes place within the hair matrix (Figs 2,3). A subcompartment of the matrix known as theprecortex is the origin of the hair-shaft lineages (Fig. 2). Analysis of chimeric follicles expressingdifferent genotypic markers has shown that, inaddition to cells whose progeny contribute to all sixIRS and hair-shaft lineages [31] (putative stem cells),there is a common matrix progenitor cell for thehair-shaft lineages and one for the IRS lineages[10,34] (Fig. 3). The ORS represents a separatelineage [34] (Fig. 3).

Whereas the matrix is agreed to be the origin of the IRS and hair-shaft lineages, the location of thecompanion layer (Fig. 2) progenitors has been more of a mystery. The companion layer has been variouslydescribed as being a component of the IRS or the ORS, or as being a completely distinct lineage[4,35,36]. Analysis of keratin production patternswithin the hair follicle lineages suggests that the companion layer is not derived from ORS progenitors [36]. Rather, the observation ofcoexpression of the transcriptional regulator CCAAT displacement protein (CDP) in both the IRSand companion cells suggests that the companioncells constitute the fourth and outermost layer of theIRS [37] (Fig. 3).

Proliferation versus terminal differentiation

Stem-cell progeny must make two decisions. The first is whether or not to remain a stem cell. Havingdecided to leave the stem compartment, a cell mustthen select a particular differentiation pathway,leading ultimately to irreversible loss of proliferativecapacity. In regulating the balance betweenproliferation and differentiation in postnatal hairfollicles, the secreted signalling molecules Sonichedgehog (Shh) and bone morphogenetic proteins(Bmps) are of central importance. Shh promotesproliferation and Bmps promote differentiation. In adult epidermis, Shh expression is restricted tocells at the distal portion of the growing follicle[38,39]; mice treated with anti-Shh antibodies showimpaired epithelial growth at sites of folliculogenesis[40]. Several Bmp family members are produced inthe postnatal hair-shaft precursor cells and in thedermal papilla [41]. Inhibition of Bmp signalling bythe Bmp antagonist Noggin is required for new hairgrowth in postnatal skin and the growth-inducingeffect of Noggin is mediated, at least in part, by Shh [42]. In transgenic mice that produce Noggin in proliferating hair matrix cells and earlydifferentiating precursors of the IRS and hair shaft,differentiation of the hair shaft but not the IRS isseverely impaired; cells in the precortex and hairshaft continue to proliferate and to express genes thatare normally downregulated during differentiation[43]. There is evidence that the homeodomaintranscription factors Msx1 and Msx2 are Bmp targets [43] and, consistent with this, overproductionof Msx2 in the hair matrix reduces proliferation andinduces premature differentiation [44].

Other factors believed to regulate the balancebetween epidermal proliferation and differentiationinclude the transcription factors Forkhead-box n1(Foxn1) [45] and nuclear factor κB (NF-κB) [46]. Inpostnatal mouse epidermis, Foxn1 is produced indifferentiating cells of the IFE and in postmitoticprecursor cells of the hair shaft and IRS, as well as incells undergoing the transition from a proliferative toa postmitotic state [45]. Loss of Foxn1 function,resulting in the nude mouse phenotype, leads to afailure to form the cortex and causes defects in theIRS and hair cuticle; the IFE also undergoes aberrantdifferentiation. Manipulation of the NF-κB pathwayby deletion or overproduction of different componentshas established roles in immune responses and inregulating the decision of epidermal cells toproliferate or differentiate [46]. NF-κB might also beinvolved in lineage commitment, because it isrequired for normal morphogenesis of hair folliclesand sweat glands downstream of the tumour necrosisfactor receptor family member EDAR [47].

Lineage selection within the hair follicle

Many transgenic and knockout mice have strikinghair abnormalities, such as hair loss or a ruffled coat[2]. These can provide clues to the identities of



188 Review

molecules that regulate the decision to embark along different hair lineages and of molecules that are required for execution of a specificdifferentiation programme. An example of the secondclass is the homeodomain transcription factorHoxc13, which is produced in cells of the hair matrixand the lower shaft of growing follicles [48,49]. In Hoxc13-null mice, hair follicle formation duringdevelopment is normal but the hair shafts fracture atthe skin surface [50]. This phenotype appears to occurbecause Hoxc13 activates the expression of severalgenes for hair keratins during early hair-lineagedifferentiation [48,49].

One molecule for which the consequences of loss ormisexpression have been well worked out is Notch, areceptor that is activated by transmembrane proteinligands such as Delta and Jagged. The research onNotch highlights the importance of direct cell–cellcontact in regulating epidermal cell fate. In mouseepidermis, Notch1 is produced in matrix cells,whereas Notch3 is produced in the precursor anddifferentiating cortex and cuticle of the hair follicle[51]. Expression of a constitutively active Notch1transgene in the cortex of the hair follicle results inabnormal differentiation of the medulla and cuticle,two neighbouring cell types that are transgenenegative [51]. Notch might either direct thedifferentiation of follicular cells or assist those cells tointerpret signals emanating from the dermal papilla[51]. Notch1 is also found in all the viable layers ofhuman IFE [32] and keratinocyte-specific inducibledeletion of Notch1 results in IFE hyperproliferation[52]. This is consistent with the observation that, incultured human IFE, activation of Notch at the edgesof stem-cell clusters promotes neighbouring cells toenter the transit amplifying compartment [32].

Another key regulator of lineage choice in the hairfollicle is Cutl1, which encodes mammalian CDP.CDP is produced in the hair bulb, IRS progenitor cellsof the matrix, the three differentiating layers of theIRS and the companion cell layer [37]. Mice with anull mutation in Cutl1 have disrupted hair folliclemorphogenesis [37]: differentiation of IRS cells isimpaired, the cell layers of the IRS are reduced andthe ORS is enlarged, possibly as a result of ectopicShh signalling. The absence of CDP does not interferewith cell proliferation within the matrix but leads toderegulated expression of IRS-specific genes in thehair shaft.

Lineage choice within the hair follicle is alsocontrolled by secreted factors and these include Wnt3and Bmp4. In growing hair follicles, Wnt3 is producedin the precursor cells of the hair-shaft medulla andalso in the IFE [53]. Overproduction of Wnt3 in theORS via the keratin14 promoter causes defects inhair-shaft structure and elevated expression ofseveral hair-shaft proteins, suggesting that one of the functions of Wnt3 is to regulate hair-shaftdifferentiation [53]. Bmp4 is activated in hair-shaftprecursor cells and in the dermal papilla [41]. Ectopic

production of Bmp4 in the ORS inhibits proliferationin the hair matrix and activates hair keratin genes inthe ORS [54].

Sebocyte differentiation

Sebaceous glands are intimately associated with hair follicles and the two structures are often referred to collectively as the pilosebaceous unit.Nevertheless, there are conditions in which hairfollicles are induced without associated sebaceousglands [7] and there are molecules that promotesebocyte differentiation independent of induction of hair lineages. One of these is the product of theproto-oncogene c-Myc, which is expressed in several subpopulations of epidermal cells, including the basal layer of the IFE, the bulge, the proliferating cells of the hair bulb and thedifferentiating matrix cells that give rise to the hair fibre [55].

c-Myc induces differentiation of human IFE stemcells into transit amplifying cells in vitro [56]. When ac-Myc transgene is expressed in the hair follicles andthe basal layer of the IFE (via the keratin14promoter), adult mice gradually lose their hair anddevelop spontaneous ulcerated lesions owing to asevere impairment of wound healing [57]. Theepidermis is hyperproliferative and analysis of label-retaining cells shows a significant depletion of stemcells [57]. Cells that are stimulated to exit thestem-cell compartment differentiate into sebocytesand IFE at the expense of the hair lineages, andtransient activation of Myc is as effective asconstitutive activation in inducing these effects [58].Given that c-Myc activation decreases integrinexpression [56,57] and inhibits keratinocytemigration [57], one explanation for the stimulation ofsebocyte differentiation is that bulge progeny areunable to move down the follicle and therefore remainin a sebocyte-conducive environment, distant fromthe dermal papilla. An unresolved issue is whetherthe stem-cell depletion effect is Myc specific orwhether other factors that stimulate stem cells toproliferate will also stimulate the production oftransit amplifying cells.

A second regulator of sebocyte differentiation isthe transcription factor peroxisome proliferator-activated receptor γ(PPARγ). PPARγfunctions as aheterodimer with the retinoid X receptor (RXR),binding to specific sites on DNA and activatingtranscription in response to ligand. PPARγ-null mice die in mid-gestation but the contribution ofPPARγto adult tissues has been examined bygenerating mice that are chimeras of PPARγ −/− andwild-type cells [59]. Analysis of the skin of these mice suggests that PPARγmight be required forsebocyte differentiation because all the sebaceousglands are derived from wild-type cells; no hairphenotype has been reported. Conversely, PPARγligands promote the differentiation of sebocytes inculture [60].



189Review

Interconversion of hair follicles and IFE

Hair loss in association with the formation ofepithelioid cysts within the dermis is observed inseveral mutant mice (Fig. 4b). For example, cystswith absent or abnormal hair shafts are observed onoverproduction of Zipro1 [61], a zinc-fingertranscription factor that is produced in hair follicles,sebaceous glands and the IFE, or after ablation ofRXRα [62] or the vitamin D receptor [63] in adultmouse epidermis.

One interpretation of the cyst phenotype is that itreflects the suppression of hair-type differentiationpathways and the induction of IFE differentiation.The outer layer of the cyst corresponds to the IFEbasal layer and the inner layers correspond todifferent stages of IFE terminal differentiation,culminating in the accumulation of cornified cells inthe centre of the cysts [64]. Whether cyst formationreflects the transdifferentiation of cells that had

previously committed to hair lineages or thereprogramming of stem cells (for example, as a resultof loss of dermal papilla signals) remains to bedetermined [64]. However, cyst formation providesclear evidence for continuous regulation of lineagecommitment in postnatal life.

The genesis of dermal cysts has been characterizedmost extensively in mice with a mutation in theHairless (hr) gene, which encodes a putativetranscription factor [4,65]. Mice with the hairlessmutation are born with hair follicles that appearnormal but suffer from postnatal hair loss. Thefollicles degenerate, leaving behind dermal cysts andstructures called utriculi that open onto the surface of the epidermis. In hr skin, the distal ORS retains its integrity and forms utriculi, whereas the rest ofthe ORS disintegrates. The remnants of the bulgebecome isolated from other epithelial cells but retaintheir capacity to proliferate and produce eithercolumnar epithelial outgrowths or dermal cysts.Cysts can also arise from the disintegrating ORS and from the base of the hair follicle. Dermal cysts inhr mice not only reflect IFE differentiation: somecysts contain sebocytes and others show evidence of residual hair differentiation. Thus, the differenttypes of cysts might reflect which parts of the ORSgive rise to them.

If cysts reflect the conversion of hair into IFE thenthe converse phenotype is de novo induction of hairfollicles in postnatal IFE. In fact, both phenotypes can be created by modulating the activity of β-catenin,a key effector of the Wnt pathway that translocates tothe nucleus to activate transcription during Wntsignalling [1,66]. Several Wnt proteins and theirreceptors are expressed in the IFE, hair follicle anddermal papilla of postnatal mice [67], together with two downstream transcription factors, Tcf3 and Lef1. Tcf3, which can act as a transcriptionalrepressor, is produced in the bulge and ORS, whereas Lef1 is produced in cells in the hair matrix(progenitors of the IRS and hair shaft) [68,69]. In addition, a subpopulation of matrix cells stainspositive with an antibody to Tcf3/4 [31]. During hairgrowth, active Wnt signalling occurs in the bulge, in the precortex cells of the matrix and in the dermal papilla, as evidenced by accumulation ofnuclear β-catenin (Fig. 4a) or reporter gene activation [64,68,69]. Wnt proteins maintain the hair follicle inductive abilities of cultured dermalpapilla cells [70].

Inhibition of Wnt signalling by ablation of theβ-catenin gene in mouse epidermis [71] or byexpressing an N-terminally truncated Lef1 (∆NLef1)transgene that lacks the β-catenin binding site [64,69]causes hair loss and the appearance of dermal cysts(Fig. 4b). Conversely, expression of stabilizedβ-catenin under the control of the keratin14 promotercauses de novo formation of hair follicles in postnatalanimals [38]. Thus, the level of β-catenin signallingdetermines lineage choice: activation of the pathway



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(b)

(a)

Fig. 4. β-Catenin signalling in postnatal epidermis. (a) Hair follicle bulbstained with an antibody against β-catenin [64], showing nuclearaccumulation in the dermal papilla (arrowheads) and precortex(arrows). Bar, 20 µm. (b) Conversion of hair follicles into cysts ofinterfollicular epidermis in transgenic mice expressing an N-terminaltruncation of the transcription factor Lef-1, which is unable to bindβ-catenin [64]. Arrows indicate residual hair shafts; arrowheads indicateclusters of sebocytes associated with cysts. Bar, 200 µm.

stimulates hair-type differentiation, whereasinhibition of the pathway promotes differentiation of IFE and sebocytes.

Remarkably, the same effect on lineage selection is observed in tumours: transgenic animalsoverproducing β-catenin develop tumours withcharacteristics of hair-type differentiation [38],whereas tumours that develop in mice expressing a∆NLef1 transgene show sebocyte and IFEdifferentiation [64]. This raises the issue of whetherperturbation of β-catenin signalling is a primaryoncogenic event or whether β-catenin interactssynergistically with additional oncogenes todetermine the type of tumour that is formed.

Concluding remarks

Here, we have considered the epidermal stem-cellcompartment and the differentiated daughter cells that it produces, and we have highlighted someof the factors that regulate the different lineages.Before closing, there are several additional points tomake that underscore the complexity of the system.First, although we have focused on postnatal

epidermis, most, if not all, of the regulators that wehave described are also important in hair follicleinduction during development [1]. It is intriguing that the same molecules can play different roles inembryonic and postnatal skin [39,43]. Second, thedifferent signalling pathways do not function inisolation but interact both positively and negatively;thus, modulating the concentration of one factor can affect signalling by another [39,67]. Finally, the temporal and spatial patterns of expression of the growth factors and their receptors can beextremely complex, with expression of multiplefamily members being detected both in themesenchyme and in distinct subpopulations ofkeratinocytes [67,72].

Although considerable progress has been made in defining lineage relationships within theepidermis, we are still a long way from completelycharacterizing all the intrinsic and extrinsicregulators of each step in the transition from amultipotent stem cell to the ten terminallydifferentiated cell types shown in Fig. 3. We see thisas the major challenge for the future.



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