getting under the skin of epidermal morphogenesis

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© 2002 Macmillan Magazines Ltd The epidermis is a thin layer of stratified squamous epithelium that rests on top of a basement membrane of extracellular matrix, which separates it and its appendages, including the hair follicles and sweat glands, from the underlying mesenchymally derived dermis (FIG. 1). The epidermis is tough and resilient, and is able to withstand the physical and chemical traumas of each passing day. It must keep harmful microorganisms out in order to guard against infection, and retain body fluids in order to prevent dehydration. Whether a fish, bird or mammal, the epi- dermal surface, in all its splendid and varied cloaks, is the source of survival and perpetuation. In this review, we discuss emerging data on how the epidermis bal- ances growth with differentiation, and how these data have increased our understanding of the genetic basis of human skin disorders. Epidermal stem cells: the secret of self-renewal Few people die because their skin epidermis has natu- rally exhausted its ability to replenish itself. And unless our skin is badly burned or cut, our epidermis can naturally heal and repair. How can it accomplish such remarkable feats? The epidermis is a self-renew- ing tissue: a single, adult skin stem cell has sufficient proliferative capacity to produce enough new epider- mis to cover the body surface 1 (reviewed in REF. 2). In the skin of furry mammals, slow-cycling, epithelial stem cells reside in a portion of the hair follicle known as the bulge (FIG. 1). These stem cells are MULTIPOTENT and can give rise not only to epidermis, but also to hair follicles and sebaceous glands 3,4 . Those stem-cell progeny that exit the bulge and migrate upwards into the epidermis populate the innermost (basal) layer. The rate of proliferation and upwards migration is greatly accelerated when the skin is injured and wound healing is induced. In human skin, which has a meagre version of the hair coat of most mammals, the epidermis is much thicker and the basal layer harbours highly proliferative self-renewing cells. This has led scientists to wonder whether this subset of epidermal cells is similar or iden- tical to the multipotent stem cells in the bulge of the hair follicle. Most researchers accept a universal definition of a stem cell as one that can divide to produce both daughter stem cells and cells that go on to differentiate. According to these criteria, a subset of basal epidermal cells (at least in human skin), as well as bulge cells, quali- fies as stem cells. Whether stem cells in the basal layer have the capacity to generate hair follicles, as bulge stem cells do, remains unclear and is a topic of considerable attention in the field. Most cells within the basal layer are the rapidly dividing progeny of stem cells 5,6 . Referred to as transit amplifying, these cells undergo a limited number of divisions before they withdraw from the cell cycle, GETTING UNDER THE SKIN OF EPIDERMAL MORPHOGENESIS Elaine Fuchs and Srikala Raghavan At the surface of the skin, the epidermis serves as the armour for the body. Scientists are now closer than ever to understanding how the epidermis accomplishes this extraordinary feat, and is able to survive and replenish itself under the harshest conditions that face any tissue. By combining genetic engineering with cell-biological studies and with human genome data analyses, skin biologists are discovering the mechanisms that underlie the development and differentiation of the epidermis and hair follicles of the skin. This explosion of knowledge paves the way for new discoveries into the genetic bases of human skin disorders and for developing new therapeutics. MULTIPOTENT STEM CELL A stem cell that has the potential to give rise to multiple cell lineages. NATURE REVIEWS | GENETICS VOLUME 3 | MARCH 2002 | 199 Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Room N314, 5841 South Maryland Avenue, Chicago, Illinois 60637, USA. Correspondence to E.F. e-mail: [email protected] DOI: 10.1038/nrg758 REVIEWS

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Page 1: GETTING UNDER THE SKIN OF EPIDERMAL MORPHOGENESIS

© 2002 Macmillan Magazines Ltd

The epidermis is a thin layer of stratified squamousepithelium that rests on top of a basement membraneof extracellular matrix, which separates it and itsappendages, including the hair follicles and sweatglands, from the underlying mesenchymally deriveddermis (FIG. 1). The epidermis is tough and resilient,and is able to withstand the physical and chemicaltraumas of each passing day. It must keep harmfulmicroorganisms out in order to guard against infection, and retain body fluids in order to preventdehydration. Whether a fish, bird or mammal, the epi-dermal surface, in all its splendid and varied cloaks, isthe source of survival and perpetuation. In this review,we discuss emerging data on how the epidermis bal-ances growth with differentiation, and how these datahave increased our understanding of the genetic basisof human skin disorders.

Epidermal stem cells: the secret of self-renewalFew people die because their skin epidermis has natu-rally exhausted its ability to replenish itself. Andunless our skin is badly burned or cut, our epidermiscan naturally heal and repair. How can it accomplishsuch remarkable feats? The epidermis is a self-renew-ing tissue: a single, adult skin stem cell has sufficientproliferative capacity to produce enough new epider-mis to cover the body surface1 (reviewed in REF. 2). Inthe skin of furry mammals, slow-cycling, epithelial

stem cells reside in a portion of the hair follicle knownas the bulge (FIG. 1). These stem cells are MULTIPOTENT

and can give rise not only to epidermis, but also tohair follicles and sebaceous glands3,4. Those stem-cellprogeny that exit the bulge and migrate upwards intothe epidermis populate the innermost (basal) layer.The rate of proliferation and upwards migration isgreatly accelerated when the skin is injured andwound healing is induced.

In human skin, which has a meagre version of thehair coat of most mammals, the epidermis is muchthicker and the basal layer harbours highly proliferativeself-renewing cells. This has led scientists to wonderwhether this subset of epidermal cells is similar or iden-tical to the multipotent stem cells in the bulge of the hairfollicle. Most researchers accept a universal definition ofa stem cell as one that can divide to produce bothdaughter stem cells and cells that go on to differentiate.According to these criteria, a subset of basal epidermalcells (at least in human skin), as well as bulge cells, quali-fies as stem cells. Whether stem cells in the basal layerhave the capacity to generate hair follicles, as bulge stemcells do, remains unclear and is a topic of considerableattention in the field.

Most cells within the basal layer are the rapidlydividing progeny of stem cells5,6. Referred to as transitamplifying, these cells undergo a limited number ofdivisions before they withdraw from the cell cycle,

GETTING UNDER THE SKIN OFEPIDERMAL MORPHOGENESISElaine Fuchs and Srikala Raghavan

At the surface of the skin, the epidermis serves as the armour for the body. Scientists are nowcloser than ever to understanding how the epidermis accomplishes this extraordinary feat, andis able to survive and replenish itself under the harshest conditions that face any tissue. Bycombining genetic engineering with cell-biological studies and with human genome dataanalyses, skin biologists are discovering the mechanisms that underlie the development anddifferentiation of the epidermis and hair follicles of the skin. This explosion of knowledge pavesthe way for new discoveries into the genetic bases of human skin disorders and for developingnew therapeutics.

MULTIPOTENT STEM CELL

A stem cell that has the potentialto give rise to multiple celllineages.

NATURE REVIEWS | GENETICS VOLUME 3 | MARCH 2002 | 199

Howard Hughes Medical Institute,Department of MolecularGenetics and Cell Biology,The University of Chicago,Room N314,5841 South MarylandAvenue, Chicago,Illinois 60637, USA.Correspondence to E.F.e-mail:[email protected]: 10.1038/nrg758

R E V I E W S

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KERATIN

A cytoskeletal filament that istypically 10 nm in diameter.

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repressing the transcription of the epidermal growthfactor receptor (EGFR) gene and genes regulated byp53 and the cell cycle11, and any of these functionscould maintain stem cells in a slow cell-cycling state.That said, mice that lack Trp63, which encodes p63,have a very thin epidermis and severe defects in epi-dermal proliferation, stratification and differentia-tion11,12. The early onset of these defects, togetherwith the normal expression pattern of Trp63 and itsnull phenotype, indicates a role for p63 in maintain-ing transit-amplifying epidermal-cell character ratherthan that of a stem cell.

By contrast, the elevated expression of the cell-cycleregulator, c-Myc (myelocytomatosis oncogene; alsoknown as Myc), in the stem and transit-amplifyingcells of transgenic mice leads to epidermal hyperprolif-eration without loss of differentiation13,14. Additionally,these animals lose their hair and have severelyimpaired wound healing, which both indicate theaccelerated use and eventual depletion of the stem-cellpopulation of the skin. Whether c-Myc is naturallyactivated as stem cells exit the bulge and convert totransit-amplifying cells is an intriguing question thatremains unaddressed, although intense c-Myc expres-sion in the bulge has recently been reported15.

Wnt signalling. The genes that encode c-Myc andcyclin D1 (Ccnd1) have both been implicated as

commit to terminal differentiation, detach from thebasement membrane and begin their trek towards the surface of the skin7 (reviewed in REF. 8). Cells thatreach the body surface are dead, enucleated, flattenedcells (squames) that are subsequently sloughed and arecontinually replaced by inner cells that move out-wards. So, the epidermis is in a constant state ofdynamic equilibrium, replenishing itself every twoweeks throughout life.

A remarkable feature of adult, human epidermalstem cells is that they can be maintained and propa-gated in vitro in primary human keratinocyte cul-tures. But finding these stem cells and studying theirproperties presents a more difficult challenge, becausesuch cultures contain a mixed population of cells(reviewed in REF. 9). Nevertheless, stem-cell characterin vitro has been correlated with the selective down-regulation of certain proteins, such as the transferrinreceptor9, 14-3-3σ (a nuclear-export protein), and thecytoskeletal KERATINS, K19 and K15.

However, most of the genes that are associatedwith skin epithelial stem cells are also expressed intheir transit-amplifying offspring, which raises thequestion of whether these markers function to main-tain stem-cell character or proliferative capacity. Anexample of this is p63 — a p53 homologue that isexpressed throughout the basal layer of theepidermis10. The p63 protein has been implicated in

SCGL

Epi

derm

isD

erm

is

SLBL

Basement membrane Sebaceous

gland

Bulge

Medulla

Cortex

Cuticle

Inner root sheath

Outer root sheath

Dermal papilla

Sweat gland

Matrix

Stratum corneum (SC)

Granular layer (GL)

Spinous layer (SL)

Basal layer (BL)

Basement membrane

Adherens junction Hemidesmosome Desmosome Focal contacts

Figure 1 | The skin and its appendages. Mammalian skin consists of theepidermis and dermis, separated by a basement membrane. The epidermis is astratified squamous epithelia that is composed of several cell layers. Resting onthe basement membrane is the basal layer (BL), consisting of proliferating, transit-amplifying cells (see text). The basal layer stratifies to give rise to differentiated celllayers of the spinous layer (SL), granular layer (GL) and the stratum corneum (SC).Also shown is a cross-section of a hair follicle, which consists of an outer rootsheath that is contiguous with the basal epidermal layer. At the bottom of thefollicle is the hair bulb, made from proliferating matrix cells. The transit-amplifyingmatrix cells terminally differentiate to generate the different cell types of the follicle.Also shown is the bulge, which is part of the outer root sheath and is whereepidermal stem cells reside. The dermal component of the hair follicle is the dermalpapilla, which consists of specialized mesenchymal cells surrounded by the hairmatrix cells.

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colleagues suggest that the role of Notch in neural-crest stem cells is to elicit a cell-heritable switch of fatein the stem cells that neighbour Notch-activatedcells26. Such a role has also been postulated for Notchin Drosophila melanogaster, in which it is cruciallyrequired for epidermal-cell-fate specification27.Whether Notch signalling has a similar role in specify-ing mammalian epidermal cell fate is not as clear. Theexpression of Notch1 occurs throughout the adult epi-dermis and in the outer root sheath and matrix cellsof the hair follicles28–30 in mice. What might Notch bedoing in these cells? In human epidermis, expressionof the Notch ligand DLL1, which encodes Delta-like 1(also called Delta1) is confined to the basal layer, andis most highly expressed where stem cells seeminglyreside31, which indicates a possible role forNotch/Delta signalling in stem-cell maintenance.However, additional Notch ligands, jagged 1 (JAG1)and jagged 2 (JAG2), are upregulated by p63 through-out the basal layer32, which indicates a role forNotch/Jagged signalling in epidermal differentiation.Consistent with this idea is the finding that the condi-tional ablation of Notch1 in mouse skin epitheliaresults in epidermal hyperproliferation and reduceddifferentiation33. If Notch signalling acts to promoteepidermal differentiation in the postnatal epidermis,then a similar function might be assigned to itsexpression in the outer root sheath of the hair follicle,which shares many similarities with the epidermis inits differentiation programme. The role of Notch inthe hair matrix, however, is more mysterious. In earlyskin development, Dll1 is expressed in the dermalcondensates that underlie sites of subsequent hair-fol-licle morphogenesis28. The expression of epithelialNotch and mesenchymal Delta indicates a possiblemechanism by which the distinction between theepithelial and mesenchymal components of the hairfollicle is achieved and/or maintained.

Intriguingly, when Notch signalling is preventedin mice by inactivating Psen1, which encodes the pur-ported Notch1-processing enzyme presenilin 1, stableβ-catenin accumulates and activates Lef1 transcrip-tion, which is typically associated with hair differenti-ation34. Although these findings indicate aninhibitory role for Notch signalling in hair formation,it is important to note that Psen1 can promote β-catenin turnover independently of Notch35–37.Additionally, other studies have shown that on activa-tion of Notch, the processed intracellular domain ofNotch becomes a transcription co-factor for Lef1-regulated gene targets, but these targets seem to bedistinct from those that are activated through β-catenin or by canonical Wnt signalling38. Thisraises the possibility that Notch signalling mightinterfere with Wnt signalling and with the specifica-tion of the hair-cell fate in matrix cells. Although fur-ther studies are required to address this point, find-ings so far indicate that the development of theepidermis and its appendages might be governed by atug-of-war between the Notch and Wnt pathways,with β-catenin, Lef1 and Notch peptide at the centre.

transcriptional targets of complexes that are involvedin the canonical Wnt (wingless-related) signallingpathway (FIG. 2; reviewed in REF. 16). Several Wnt genesare expressed in the skin17,18. An enhancer that isresponsive to the Wnt-signal-transduction complex of β-catenin and Lef1 (lymphoid enhancer bindingfactor 1) has been shown to drive the expression of alacZ reporter gene in both the multipotent ectodermand its underlying dermal condensates during skin development19. So, Wnt signalling and nuclear β-catenin/Lef1-mediated changes in transcriptionoccur coincidentally in embryonic ectoderm with thecommitment of embryonic ectoderm to adopt a hairfollicle, rather than an epithelial, fate19.

That Wnt signalling is functionally important forhair-follicle morphogenesis has been shown in sev-eral ways20–23. At least two members of the Lef1/Tcf(T-cell factor) family of DNA-binding proteins areexpressed in skin. Lef1 is preferentially expressed inthe precortex of the hair follicle, where, in conjunc-tion with β-catenin, it seems to activate hair-specifickeratin gene expression24. By contrast, Tcf3 (T-cellfactor 3) is preferentially expressed in the bulge stem-cell compartment, where it seems to act as arepressor protein, perhaps to repress c-Myc or cyclinD1 (REF. 21). Immunohistochemical studies indicatethat when stem cells exit this compartment and pop-ulate the sebaceous gland and epidermis, they loseTcf3 expression, but if they populate the matrix of thehair follicle, they replace Tcf3 with its transactivatingcousin Lef1 (REF. 21). Although Tcf3 has not yet beenablated in mice, its expression has been driven in thebasal cells of transgenic mice under the control of abasal epidermal keratin (keratin 14; also known asKrt1-14) promoter. This expansion of Tcf3 expres-sion, beyond its normal expression domain, results inthe repression of epidermal differentiation and thegeneration of cells with the biochemical profile ofouter-root-sheath and stem-cell compartment cells,which are normally positive for Tcf3 (REF. 21).Moreover, when the coordinate action of Tcf3/Lef1and β-catenin is blocked, either through the condi-tional ablation in mice of Catnb, which encodes β-catenin, or through the transgenic expression of amutant Lef1 that lacks the β-catenin-binding site,hair-follicle differentiation is impaired and cells dif-ferentiate along epidermal and/or SEBOCYTE lin-eages21–23. Taken together, these fascinating pheno-types indicate that β-catenin/Lef1-mediated genetransactivation is required for hair-follicle morpho-genesis, whereas interference with this pathwayand/or the relief of Tcf3-mediated repression mightbe required for basal epidermal-cell specification.

Notch signalling: to be or not to be epidermisAnother signalling pathway that might stimulate stemcells to adopt an epidermal fate is the Notch signallingpathway. The Notch family of cell-surface receptorsuses several different ligands that signal in either apositive or negative manner to alter gene expression invarious developmental programmes25. Anderson and

SEBOCYTE

A cell of the sebaceous gland.

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premature differentiation can yield a thin epidermis, asseen in sun-damaged or aged skin. The epidermis con-trols this balance in part by orchestrating a transcrip-tional programme that produces temporally and spa-tially distinct epidermal cells, each able to carry outtasks that are specific to their position within the skin.

As cells withdraw from the basal layer, they stopdividing and induce a programme of terminal differ-entiation that will ultimately allow them to function asthe barrier for the skin. Although much is knownabout the patterns of expression of structural genes inthe epidermis and its appendages, much less is knownabout how these patterns are established during devel-opment and how programmes of terminal differentia-tion are orchestrated at the transcriptional level. Invitro studies have been instrumental in identifyingtranscription factors, such as basonuclin, that stimu-late rRNA gene expression and help sustain prolifera-tion42, and factors such as C/EBP43, the POU domaintranscription factors Oct6 (REF. 44) and Oct11 (REF. 45),and ESE2 (also known as ELF5; an ETS domain tran-scription factor)46, Klf4 (Kruppel-like factor 4)47, andthe retinoic acid receptors (RARs) and their partners48,which are preferentially found in the terminally differ-entiating cells of the epidermis. The inactivation ofgenes that encode epidermal transcription factors bygene targeting has now begun to reveal importantfunctional information about these factors and theirrole in human genetic skin disorders.

Antagonism between signalling pathways also takesplace during hair morphogenesis. Sonic hedgehog (Shh)signalling has been shown to promote the proliferationof the hair precursor (matrix) cells, and noggin, a nega-tive regulator of bone morphogenetic proteins (BMPs),promotes activation of Lef1, which subsequently pro-motes hair differentiation upon Wnt signalling(reviewed in REF. 39). Shh and the BMPs also have a rolevery early in embryogenesis as cells choose between aneural and ectodermal fate: BMP signalling promotesectodermal development, whereas Shh promotes NEURAL

INDUCTION40. Neural induction also requires signalling byfibroblast growth factors (FGFs). Interestingly, in thechick embryonic ectoderm, the state of WNT signallingis a key determinant of neural and epidermal cell fates41.Continual WNT signalling blocks the response ofEPIBLAST CELLS to FGF signals, allowing the expression ofthe BMPs, and signalling through the BMP pathway, todirect epiblast cells to an epidermal cell fate41. The com-plex interplay between Shh, BMPs and FGFs, and per-haps their regulation by Wnts, might therefore act to dis-tinguish epidermal, and neuronal cell fates.

Epidermal development and transcriptionGrowth and differentiation are tightly linked processesin the epidermis, which need to be precisely balanced. Ifthere are too many dividing cells, hyperproliferativedisorders of the skin can result, such as psoriasis andbasal- or squamous-cell carcinomas. Conversely,

NEURAL INDUCTION

The specification of cells thatgive rise to the neural tube and,ultimately, to the central nervoussystem.

EPIBLAST CELL

A cell in the early embryo thatgives rise to all three definitivegerm layers of the embryo: theectoderm, mesoderm andendoderm.

Notch signalling

Dsh (off)β-cat

β-catenin

degraded

E-cadherin

Gbp

Gsk3

Axin

β-cat

Apc

P

β-cat

Lef1 Targets

Nucleus

Notch signalling

?

Dsh (on)β-cat E-cadherin

Frizzled Frizzled

Gsk3

Axin

β-catβ-cat

β-cat

β-cat

Apc

P

Gbp

Lrp

Lrp

Wnt

WntsFRPs

Dkk2

Lef1 Targets

Nucleus

a Unstimulated (epidermal cell) b Stimulated (hair cell)

Figure 2 | The Wnt signalling pathway in skin. a | In a Wnt-unstimulated cell, Gsk3 phosphorylates the cytoplasmic pool of β-catenin (β-cat) and promotes its degradation through a ubiquitin-mediated proteasome pathway. Lack of Wnt stimulation isthought to promote epidermal cell fate. b | In a Wnt-stimulated cell, the Wnt receptor frizzled activates dishevelled (Dsh), which inturn inhibits Gsk3 activity. On its inhibition, Gsk3 no longer phosphorylates β-cat, and so β-cat begins to accumulate in the cytosol.Stable β-cat subsequently enters the nucleus, forms a transcriptional complex with members of the Lef1/Tcf family of DNA-bindingproteins and regulates downstream target genes. The interaction between Wnts and their receptors is also modulated by otherextracellular proteins, such as secreted frizzled-related proteins (sFRPs), and dickkopf2 (Dkk2), as well as transmembrane co-receptor proteins, such as LDL-receptor-related protein1 (Lrp1). Apc, adenomatous polyposis coli; Gsk3, glycogen synthasekinase 3β; Lef1, lymphocyte enhancer binding factor 1; Tcf, T-cell factor.

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Conditional gene targeting in mouse skin of the Rxra(retinoid X-receptor alpha) gene, which encodesRxrα, provided additional evidence that epidermalhyperplasia and aberrant terminal differentiationoccur when Rxrα function is compromised in theskin47. Rxra mutant mice also have alopecia, a featurethey share with hairless63 and vitamin D receptormutant mice47. A role for retinoid receptors in pro-moting epidermal differentiation is difficult to recon-cile with the known proliferative effects of topicallyapplied retinoids on skin epidermis. Sifting throughthese and other potential complexities must wait forthe generation of multiple conditional mouse knock-outs of various RXR partners in the skin.

Other regulatory proteins in epidermal development.Several other transcription factors have been impli-cated in epidermal growth and differentiation. Someof these factors, such as Klf4, had not been previouslyimplicated in epidermal transcription, and it was onlythrough gene targeting that this factor was discoveredto have a central role in the late stages of epidermal dif-ferentiation40. Other factors, such as AP2, Sp1 (trans-acting transcription factor 1) and AP1, were uncoveredin searches for functional transcription-factor bindingsites in promoters, and for enhancers of genes that areexpressed largely, if not solely, in skin epidermis64–72.Like the retinoid receptor family, the potential func-tional redundancy between AP2, Sp1 and AP1 familymembers has made it difficult to explore their func-tions in skin epidermis. However, wherever ker-atinocyte-specific protein binding has been mapped toepidermally expressed genes, sites for AP2, Sp1 and/orAP1 have frequently been found73,74 (C. K. Kaufmanand E.F., unpublished observations). Moreover, severalAP2, Sp1 and AP1 family members are expressedprominently, and often differentially, in both dividingand differentiating cells of the epidermis57,66,67. It is acurious fact that AP2, Sp1 and AP1 family membersseem to be involved both in the expression of genes inthe basal layer of the epidermis, such as those for keratins 5 (KRT5) and 14 (KRT14), and in the expres-sion of keratins 1 (KRT1) and 10 (KRT10), and theCORNIFIED ENVELOPE proteins, which are present in termi-nally differentiating keratinocytes74–76.

Sorting through the AP2, Sp1 and AP1 family mem-bers to find their possible functions in epidermal-spe-cific gene expression is a painstaking and continuingprocess. Studies of AP1-regulated genes in the skin arecomplicated by the fact that AP1 transcription factorsare influenced by intracellular signalling, which can betriggered by various external stimuli, including growthfactors, cytokines and calcium (for a review, see REF. 77).Such stimuli can alter both the levels and the activity ofAP1 binding factors68, often yielding varying resultswhen promoter activities are examined in keratinocytesin vitro or in vivo. Additionally, an increased resistanceto skin tumorigenesis is seen in mice that lack either theAP1 member c-fos (FBJ osteosarcoma oncogene), orMapk9 (mitogen-activated protein kinase 9), whichencodes the Jun kinase 2 protein that phosphorylates

The NF-κB connection. The recent use of geneticengineering in mice to uncover the basis of humanskin disorders is well exemplified by knockout studiesof the I kappa kinase (IKK) complex. IKKα is a kinasethat has previously been implicated, together with itsregulatory subunit IKKγ (encoded by Ikbkg), in thephosphorylation and destruction of IκB, which is acytoplasmic inhibitor of the nuclear transcription fac-tor NF-κB. The targeted inactivation in mice of Chuk,which encodes IKKα, gave an unexpected phenotype— an epidermis that was markedly impaired in termi-nal differentiation49–51. Interestingly, the epidermaldefects that result from the loss of IKKα do not seemto be dependent on NF-κB, the activity of which isunaffected in keratinocytes that are cultured fromthese knockout animals52. Instead, IKKα seems to beresponsible for the production of a secreted factorthat promotes the differentiation of epidermal ker-atinocytes52. Just what this factor is and how it mightfit into the complex mechanisms that govern terminaldifferentiation remains, for now, a mystery.

The jury is still out as to whether NF-κB has adirect role in epidermal homeostasis. IKKβ, which ishighly homologous to IKKα, also uses IKKγ to phos-phorylate and target IκB for destruction, and this, inturn, leads to the activation of ΝF-κB. Female micethat are heterozygous for a disruption in the X-linkedIkbkg gene, show keratinocyte hyperproliferation, skininflammation and increased apoptosis. Because thephenotype of these animals closely resembles symp-toms of the X-linked skin disorder incontinentia pig-menti, it was soon discovered that patients with thisdisorder have mutations in IKBKG and aberrantIKBKG expression53–55. These findings clearly showthat compromising NF-κB function perturbs the bal-ance between epidermal growth and differentiation.However, because NF-κB has been implicated in thedifferentiation of both epidermal cells56 and immuneresponse cells, and because both are abundant in theskin, it is not yet clear whether NF-κB elicits its effectsindirectly or directly on epidermal homeostasis.

The role of retinoids. Topically applied retinoids havelong been used to treat psoriasis and precancerouslesions of the skin; however, they have a proliferativeeffect on normal skin epidermis. So, although retinoidsaffect epidermal homeostasis, their underlying mecha-nisms have proved to be difficult to untangle. Since theearly 1980s, it has been known that retinoids act at themRNA level to inhibit many aspects of differentiationand promote proliferation57,58. The discovery of thenuclear RARs and their heterodimeric partners, retinoidX receptors (RXRs), uncovered obvious candidatemediators of these effects59,60.

The multiplicity of the retinoid superfamily ofreceptors and their broad expression has made it chal-lenging to unmask the functional significance of thesereceptors in the skin by gene targeting. Transgenicmice that express dominant-negative retinoid recep-tors in skin epidermis show an impaired epidermalbarrier and suppressed epidermal differentiation61,62.

CORNIFIED ENVELOPE

An extremely tough protein lipidpolymer structure that formsjust below the cytoplasmicmembrane of cells of thecornified layer.

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be achieved through the combinatorial action of differ-ent members of these transcription factor familiesand/or through the indirect binding of keratinocyte-specific cofactors to these more broadly expressed DNA-binding proteins. The analyses of two different enhancerelements in Krt14 have revealed that, although, individ-ually, each element shows a substantial degree of ker-atinocyte specificity, neither alone could properly targetreporter gene expression to the outer root sheath andbasal layer of the epidermis, where Krt14 is normallyexpressed. In combination, however, the two elementsgave a faithful pattern of Krt14 enhancer activity73,74.Such intricacies of differentiation-specific gene expres-sion are only revealed in vivo, where keratinocytes candifferentiate along several pathways. Given the complex-ities of epithelial cell lineages in the skin, it might bequite some time before the unfinished threads of thecloak of epidermal gene regulation are fully tied.

Epidermal architecture and cell adhesionIntercellular adhesion. The epidermis is organized intoa three-dimensional lattice of tightly adhering cells. Thecellular architecture of the epidermis is essential for itsbarrier and protective functions, and this architecture isperturbed in several human genetic skin disorders,including degenerative blistering diseases, such as epi-dermolysis bullosa simplex (EBS) and palmoplantarkeratoderma, as well as in epithelial skin tumours andcancers. In the metabolically active layers of the epider-mis, intercellular adhesion is accomplished by twotypes of intercellular junction: the desmosome and theadherens junction (FIG. 3). Both of these intercellularjunctions are essential for epithelial sheet formation83,84.Recently, it has been found that the core components ofthe adherens junction, its transmembrane anchor (E-

CADHERIN) and its indirect link to the ACTIN CYTOSKELETON

are used in the first step of intercellular adhesion todraw epithelial cells together84. In the second step,desmosomal cadherins and their indirect associationwith the keratin INTERMEDIATE FILAMENT CYTOSKELETON arerequired to clamp this work into place, so that redi-rected actin polymerization can then seal the mem-branes to make the adhering sheets of cells that formthe epidermal barrier84. Consistent with this idea arethe recent findings of Runswick et al.85, who haveshown that, in vivo, both desmosomes and adherensjunctions are involved in morphogenesis and cell posi-tioning in epithelial tissues.

The formation of epithelial sheets is a basic andessential feature of the skin epidermis. It is a highlydynamic process.As the sheets migrate upwards towardsthe skin surface during terminal differentiation, epider-mal cells must constantly change their intercellular inter-actions, and, during wound healing, epidermal cellstransiently downregulate intercellular adhesion con-comitant with an increase in cell proliferation. In certaincancers, such as skin and breast cancers, similar changesalso take place, albeit in a less organized fashion.

The association between defects in adherens junc-tion proteins and human cancers is well established(reviewed in REF. 86). Most notable is the link between

AP1 family members. But defects in the balance of nor-mal epidermal growth and differentiation have not beenobserved in mice in which AP2, AP1 or Sp1 familymembers have been inactivated by gene targeting78–81.With the likely redundancy between members of thesefamilies82, it will be a long and slow process to generatethe double and/or triple knockout and conditionalknockout mouse mutants that are required to elucidatetheir precise roles in epidermal homeostasis.

The broad and diverse tissue patterns of Sp1, AP2and AP1 family members have led skin biologists tospeculate that keratinocyte-specific transcription might

E-CADHERIN

A homophilic cell-adhesionmolecule that is an importantcomponent of the adherensjunctions.

ACTIN CYTOSKELETON

A microfilament network thatconsists of filaments that are 6 nm in diameter and made upof polymerized actin. The actincytoskeleton forms the maincomponent of the cellularcontractile machinery.

a Adherens junction

b Desmosome

Plasma membranes

Extracellular

F-actin

α-catenin

β-catenin

E-cadherin

p120ctn

Vinculin

Desmoplakin

Keratin IF

Desmocollin

Desmoglein

Plakoglobin

Plakophilin

α-actinin

VASP

Figure 3 | Adherens and desmosome junctions in the epidermis. Simplified models of a | an adherens junction and b | a desmosome, which highlight some of the main protein–proteininteractions found in these structures. Adherens junctions and desmosomes mediate cell–cellcontact between all cells of the epidermis and are present in all metabolically active cell layers.The adherens junctions form a bridge between the actin cytoskeleton of neighbouring cells. Bycontrast, desmosomes associate with the keratin filament cytoskeleton of cells. Keratin IF, keratinintermediate filaments; p120ctn, adherens junction protein p120; VASP, vasodilator-stimulatedphosphoprotein. Part a modified with permission from REF. 123. Part b modified with permissionfrom REF. 83.

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The α6β

4-integrin also binds laminin 5 and is even

more crucial for epidermal attachment to the basementmembrane than is α

1-integrin (REFS 99–101). However,

in contrast to the ablation of Itga3 and Itgb1, the loss ofα

6- or β

4-integrin does not visibly compromise base-

ment membrane integrity. Instead, α6β

4-integrin forms

the core of the hemidesmosome junction, which is mor-phologically but not biochemically analogous to a half-desmosome. Hemidesmosomes associate with proteinsthat link to the keratin intermediate filament cytoskele-ton (FIG. 4). These features provide robust mechanicalstrength to the base of the skin epidermis, as well asstrong anchorage to the basement membrane102. Bycontrast, α

1-integrins associate with proteins that link

them to the actin cytoskeleton. The ability of αβ1-inte-

grins to regulate actin–myosin-driven processes, such ascell migration and movement, might explain why abla-tion of Itgb1 in mouse skin results in defects in hair-follicle formation, which requires both remodelling ofthe basement membrane and the migration of cells intothe underlying dermis.

A combination of classical human genetics andgene-targeting technology has revealed the unfortunateconsequences of mutations in the genes that encode theepidermal integrins and their associated proteins. Forexample, defects in the genes that encode the laminin 5chains were the first to reveal the relationship betweenthe devastating human blistering skin disorder junc-tional epidermolysis bullosa (JEB), and defects inhemidesmosomes and their associated components(reviewed in REF. 103). Lesions in the genes that encodeα

6- and β

4-integrins, and an associated transmembrane

protein, called BPAG2 (also known as COL17A1; colla-gen, type XVII, alpha 1) (FIG. 4), are all known to result inthe loss of hemidesmosomes, which causes the epider-mis and the underlying basement membrane to sepa-rate, as in JEB. By contrast, defects in the BPAG1e (bul-lous pemphigoid antigen 1) or plectin 1 proteins thatanchor the intermediate filament network to thehemidesmosome cause the basal epidermal cells tobecome mechanically fragile, which leads to their rup-ture on exposure to physical stress104,105.

What does the marked increase in our knowledgeof the genetic bases of these severe degenerativehuman skin disorders mean for the patient? Willthere be new and improved methods for treatingthese devastating diseases? For many of them, the sit-uation remains relatively bleak, as most are autoso-mal dominant and involve disorganized, insolublecytoskeleton. For disorders such as EBS, we can hopethat future improvements in homologous recombi-nation technologies will allow scientists to remove orinactivate the defective keratin gene from the ker-atinocytes of these patients. If such technologiesbecome possible, then natural selection, favouringthe survival of healthy basal keratinocytes at theexpense of degenerating ones, should take over andresult in a significant improvement for the patient. Ofcourse, genetic identification of the defective keratingene is now possible, affording genetic counsellingfor future EBS parents who elect to take this route.

familial colon cancer and mutations in the APC (ade-nomatous polyposis coli) gene, which encodes a pro-tein required for β-catenin turnover87. Stabilizingmutations in the β-catenin protein itself can also leadto human tumours, including a skin tumour called apilomatricoma88. Mutations in cadherins and in α-catenin have also been implicated in several differenttypes of human epithelial cancer, including lung andovarian cancers, and a possible reduction in adherensjunction proteins has been noted in squamous cellcarcinomas of the skin89,90.

Recently, it was discovered that the inverse correla-tion between intercellular adhesion and proliferationmight in part be controlled by α-catenin, whichrecruits the actin cytoskeleton to adherens junctioncomplexes91,92. When Catna1, which encodes α-catenin, is conditionally ablated in the skin of mice,their epidermal cells become hyperproliferative andshow signs of the sustained activation of theRas–MAPK (mitogen-activated protein kinase) path-way93. These findings indicate that the modification ofα-catenin — for example, by phosphorylation or byprotein–protein interactions — might transiently takeit out of commission under conditions such as woundhealing or development, when linking adhesion andproliferation might be important. It is noteworthy thatwhereas defects in adherens junction proteins are oftenimplicated in human cancers, perturbations in desmo-somal proteins are typically associated with degenera-tive disorders, including striate palmoplantar kerato-derma and ectodermal dysplasia94,95.

Basement membrane: maintaining the epithelia. Theepidermis is non-vascularized and receives its nutri-ents from blood vessels in the underlying dermis. Theepidermis and dermis are separated by a basementmembrane that is composed of extracellular matrixproteins, including collagen IV, fibronectin andlaminin 5. Both the epidermis and the dermis con-tribute to the synthesis of basement membrane com-ponents, but the basal layer of the epidermis seems tobe the sole manufacturer of collagen IV and laminin5. The basal layer of epidermal cells both synthesizesthese components and adheres to them, and also poly-merizes and/or organizes them into the basementmembrane. This has been well shown in gene-target-ing studies in mice in which Itga3 and Itgb1, whichencode the epidermal α

3- or β

1-INTEGRINS, respectively,

have been inactivated96–98. Clusters of these het-erodimeric transmembrane proteins act as laminin 5receptors, and are required for the assembly of thebasement membrane and for the adherence of epider-mal cells to it. In the epidermis, β

1-integrin also part-

ners with α2-integrin to make a receptor and orga-

nizer for collagen, and with α5-integrin to make a

receptor and organizer for fibronectin. So, in the inte-grin knockout mice, the severity of basement mem-brane synthesis or assembly defects and of the skinblistering due to subsequent epidermal detachment ismuch greater when β

1-integrin is missing than when

α3-integrin is absent96–98.

INTERMEDIATE FILAMENT

CYTOSKELETON

A network that consists offilaments, typically 10 nm indiameter, that contributes to themechanical strength of cells.

INTEGRIN

A transmembrane protein thatfunctions as a heterodimer andis involved in cell–cell andcell–extracellular-matrixinteractions.

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A myriad of evidence has underscored the impor-tance of tyrosine kinase growth-factor receptors inorchestrating the balance between proliferation and dif-ferentiation in the epidermis. Adjacent to the dermis,basal cells are exposed to a Pandora’s box of new envi-ronmental influences that distinguish these cells fromtheir differentiating counterparts. Although it has beenknown for many years that epidermal keratinocytes aredependent on mesenchymal stimuli, the molecularmechanisms that are involved are only now beginningto be discovered. Recently, it has been shown that inter-leukin 1, which is produced and released by basal epi-dermal cells, activates c-jun in the underlying dermalfibroblasts. The fibroblasts respond by producing andsecreting granulocyte macrophage colony stimulatingfactor (GM-CSF; also known as CSF2) and fibroblastgrowth factor 7 (FGF7), two factors that stimulate theirrespective tyrosine kinase receptors, and promote theproliferation and differentiation of overlying epidermalkeratinocytes109. Although neither GM-CSF nor itsreceptor are essential paracrine factors for epidermaldevelopment and/or regeneration110,111, loss of the FGFreceptor isoform Fgfr2-IIIb in the epidermis, whichbinds the dermal ligands Fgf7 and Fgf10, results ingrossly impaired epidermal proliferation in geneticallyaltered mice112. The skin phenotype of Fgfr2-IIIb-defi-cient animals is markedly more severe than that ofeither Fgf7 or Fgf10-null mice104,113. This reflects theredundancy between the FGFs and the complex natureof the external signals that must be transmitted to, andprocessed by, the epidermis to control the intricate bal-ance between epidermal growth and differentiation.

In addition to paracrine growth signals, epidermalkeratinocytes make a plethora of autocrine growth fac-tors that stimulate tyrosine kinase receptors on the sur-face of the keratinocyte. The best known of these istransforming growth factor-α (Tgf-α), a ligand for theepidermal growth factor receptor (Egfr). Mice that har-bour a null mutation in Egfr show strain-dependentdefects in epidermal (as well as hair-follicle) differentia-tion114–118. Egfr and its downstream Ras–MAPK pathwayhave also been implicated in epithelial tumorigenesis,and transgenic mice in which this pathway is constitu-tively activated develop skin papillomas107,119–121.Interestingly, transgenic mice that express a constitutivelyactive form of Son of sevenless (Sos), which is essentialfor Ras activation, develop skin papillomas with 100%penetrance, but tumour formation is inhibited and ker-atinocyte survival is impaired when these mice are bredonto a null Egfr background122. These findings reveal thatthe essential functions of Egfr in the skin go beyond itsability to activate Ras, and that it also acts as a survivalfactor in oncogenic transformation. Although it is wellknown that tyrosine kinase receptors can activate phos-phoinositol 3 kinase (PI3K) and the Akt (thymoma viralproto-oncogene) cell-survival pathway, these findingsprovide compelling functional evidence that such path-ways are also important in controlling epidermal home-ostasis. These findings also underscore the importance ofinvestigating Egfr as a target for therapeutic interventionin epithelial tumours, such as those of the skin.

For recessive disorders, such as lamellar ichthyosis(fish-like scaling) or JEB, the future seems morepromising, and in vivo functional restoration of ker-atinocytes from such patients has already beenachieved using gene therapy106.

Tyrosine kinase receptors in epidermis The ability of basal epidermal cells to adhere to theirunderlying basement membrane bestows them withfeatures that set them apart from the cells in the ter-minally differentiating suprabasal layers. Cells in thebasal layer express genes that enable them to prolifer-ate. The expression of integrins and the attachmentof basal cells to the basement membrane are requiredfor keratinocytes to activate the Ras–MAPK (mito-gen-activated protein kinase) pathway, which isinvolved in cell proliferation107. Cells can looselyattach to the basement membrane in the absence ofβ

1- or α

4-integrins, and cell proliferation still

occurs until the basement membrane disinte-grates97,98,108. These findings indicate that Ras–MAPKactivation can occur in cells as long as they areadhered to a substratum.

Figure 4 | A hemidesmosome junction. This structure isbased on biochemical and molecular evidence ofprotein–protein interactions in the hemidesmosome. α6β4-integrin heterodimers form the core of the hemidesmosome,along with BPAG2, a transmembrane protein with anextracellular domain similar to collagens. BPAG1e and plectinare two hemidesmosomal proteins that are members of theplakin family of coiled-coil proteins. These two proteins haveintermediate (keratin) filament-binding domains on their non-helical carboxy-terminal (C) segments. They concentrate onthe inner plate of the hemidesmosome, and seem to functionby linking the keratin intermediate filament cytoskeleton to thetransmembrane proteins in the hemidesmosome. BPAG1e,bullous pemphigoid antigen 1, epidermal isoform; BPAG2,bullous pemphigoid antigen 2.

β4-Integrin

IV

C

C

Anchoring filaments

Plectin

BPAG1e

BPAG2

Basement membrane

Keratin intermediate filaments

Plasma membrane

III

II

I

α6-Integrin

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Another important area for further investigationis into the factors that regulate the balance betweenepidermal growth and differentiation. The recentdiscovery that the serine/threonine kinase IKKα reg-ulates the production of a differentiation-inducingfactor for the epidermis is intriguing, but what is thisfactor, and how is IKKα regulated in the epidermis?Does IKKα stimulate the production of GM-CSF,recently implicated in keratinocyte differentiation122,or does it perhaps elicit a change in intracellular cal-cium, long known to enhance keratinocyte differen-tiation? Given the strong similarities between theIKK phosphorylation recognition sequence of IκBand β-catenin, it is tempting to speculate that IKKαmight influence epidermal differentiation by inhibit-ing the accumulation of cytoplasmic β-catenin,which is required for the differentiation of the hair-follicle lineage. However, much work needs to bedone before the true role of IKKα in skin can beunderstood.

The past couple of years have shed valuable lighton the presence of stem cells in the skin, but theproperties of these cells remain largely unknown.How do multipotent skin epithelial stem cells differfrom the multipotent stem cells of other tissues? Howdo the stem cells in the basal layer of the epidermisdiffer from those in the follicle bulge? Are both popu-lations truly multipotent, as indicated by the folliclerepopulation studies of Reynolds and Jahoda112 orcan cells in the basal layer only differentiate along theepidermal pathway? And how is the transition from astem cell to an epidermal transit-amplifying cellorchestrated? We need to answer these questions tounderstand stem-cell maintenance and lineage deter-mination. And, in turn, this information will beimportant to the issue of whether epidermal stemcells will have the clinical potential to be coaxed alongdifferentiation pathways that they do not normallytake — for example, to become a pancreatic islet cellfor the treatment of diabetes or a neuron for thetreatment of Parkinson disease.

Let us close by returning to the issues of structureand function in the skin and to the underlyinggenetic basis of skin disorders. It is surprising thatdespite nearly 20 years of molecular genetics and itsapplication to skin biology, we still know very littleabout the molecules and pathways that are involvedin the skin acquiring the characteristics of a barrier— the quintessential purpose of the epidermis. Weknow the main lipids involved49,125 and we are begin-ning to learn which transcription factors govern thelate stages of this process. However, at present, manyof the direct targets of these transcription factorsremain to be elucidated, and the precise steps in certain stages of this process, such as lamellar pro-duction and lipid secretion, are largely unsolved.As we face these new challenges and begin to unravel the mysteries still kept secret beneath thebeauty of the skin, new insights will emerge into not only the biology of the skin, but also its geneticdiseases.

Given that there are at least 20 cell types in the skin,the epidermis has many neighbours that can transmitexternal stimuli to it. The human and mouse genomesare now uncovering many new growth factors, some ofwhich have already been shown to have an effect onskin epithelia. One such example is interleukin 20(IL20), a novel IL10 homologue, which when overex-pressed in transgenic mice causes neonatal lethalityand skin abnormalities, including aberrant epidermaldifferentiation123. Expressed in skin epidermis, the IL20tyrosine kinase receptor directly phosphorylates andstimulates the nuclear STAT3 protein — a potenttransactivator of epidermal keratinocytes. Mice thatlack Stat3 in skin epithelia have profound defects inwound healing and in the hair-formation cycle124. Nodoubt, as more skin biologists delve into the wealth ofinformation that is provided by genome databases,there will be plenty of new keratinocyte factors tostimulate growth of the field for many years to come.

Conclusion and future prospectsEpidermal biologists have long recognized the impor-tance of the environment and of mesenchymal inter-actions for the establishment and maintenance of theepidermis. Recent genetic and cell-biological studiesnow underscore the importance of cell adhesion,basement membrane assembly and tissue architecturein contributing to the proper balance and spatialarrangement of growth and differentiation in the epi-dermis. During the past ten years, an elegant blend ofclassical genetics and REVERSE GENETICS has helped toreveal the underlying bases of many human skin dis-orders (see ONLINE TABLE 1). These disorders rangefrom blistering skin diseases, such as EBS and JEB, toproliferative skin disorders and cancers, includingbasal cell carcinoma and pilomatricoma. Theseadvances are an important first step to developingnew and improved methods for the diagnosis andtreatment of skin diseases. Although many importantquestions remain unaddressed in the realm of skinepithelial biology, the power of genetics, coupled withthe ancient origins of the epidermis, afford us manynew tools and model systems with which to explorethese questions.

We still know very little about the mechanisms thatactively specify and maintain epidermal cell fate, orabout the precise signalling pathways that are involvedin mesenchymal–epithelial interactions during epi-dermal growth and differentiation. Studies over thepast ten years have implicated various cell-signallingpathways, including the Notch, Wnt, FGF, Bmp, Shh,retinoic acid and TGF-β pathways in the tug of warbetween epidermal- and hair-follicle cell fates.Nevertheless, despite recent insights into the seem-ingly opposing roles of these signalling pathways, it isstill not clear how they are involved and how theyintersect in coordinating this central choice in skindevelopment. These areas are now a focus for researchin the field, and answers will undoubtedly emergewith the flurry of new experiments being conductedat present.

REVERSE GENETICS

A genetic analysis that proceedsfrom genotype to phenotype bygene-manipulation techniques,such as homologousrecombination in embryonicstem cells.

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AcknowledgementsWe thank R. DasGupta, B. J. Merrill and C. Jamora for their criticalreading of this review and for their valuable suggestions.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkAkt | APC | Apc | basonuclin | β-catenin | BPAG1e | BPAG2 |Catna1 | Ccnd1 | C/EBP | c-fos | c-Myc | Dkk2 | DLK1 | Dll1 |EGFR | Egfr | ESE2 | FGF7 | Fgf7 | Fgf10 | GM-CSF | hairless |IκB | IKBKG | IKKγ | IL10 | IL20 | Itga3 | Itgb1 | JAG1 | JAG2 |K19 | Klf4 | KRT1 | KRT5 | KRT10 | KRT14 | Krt1-14 | Lef1 |Lrp1 | Mapk9 | NF-κB | Notch (fly) | Notch (mouse) | Notch1 |p53 | p63 | p120ctn | plectin 1 | Psen1 | Shh | Sp1 | STAT3 |Stat3 | Tcf3 | Tgf-α | transferrin receptor | Trp63 | VASP | vitamin D receptorOMIM: http://www.ncbi.nlm.nih.gov/Omimepidermolysis bullosa simplex | incontinentia pigmenti | junctional epidermolysis bullosa | lamellar ichthyosis |palmoplantar keratoderma | Parkinson disease | striate palmoplantar keratoderma Mouse Genome Informatics: http://www.informatics.jax.orgRxra

FURTHER INFORMATIONDermWeb — dermatology links and resources:http://www.derm.ubc.ca/Encyclopedia of Life Sciences: http://www.els.netIntegrins: signalling and disease | Signal transduction pathways indevelopment: Wnts and their receptorsThe Wnt gene homepage:http://www.stanford.edu/~rnusse/wntwindow.htmlAccess to this interactive links box is free online.