adult corneal epithelium basal cells possess the capacity...

INTRODUCTION

In mammals, the pluristratified epithelial components of theskin and the cornea which share a common ectodermalembryonic origin have clear differences. Corneal epitheliumdoes not possess the granular cell layer and anucleate stratumcorneum typical of epidermis, and they are characterized bythe expression of different pairs of keratins: K3/K12 forcorneal epithelium and K1-2/K10 for the epidermis (Sun et al.,1983; O’Guin et al., 1987). Furthermore, only the epidermisforms cutaneous appendages such as glands and hair follicles,which express their own set of keratins (Moll et al., 1982; Heidet al., 1986).

In both adult epidermis and corneal epithelium, keratinocytepopulations continuously regenerate from stem-cellprogenitors that lead, through the combined processes of

proliferation and differentiation, to terminally differentiatedcells (Potten and Morris, 1988). It is widely accepted that stemcells are a population of slow-cycling cells that are usuallyfound in well-protected areas, and are often located close to apopulation of rapidly proliferating transient amplifying (TA)cells (among others; Lavker and Sun, 1982; Morris et al., 1985;Barrandon and Green, 1987). Epidermal stem cells are notrandomly distributed within the basal layer, but have apatterned distribution that varies with body site: for example,in the scalp they overlie the dermal papillae, whereas in thepalm they lie at the tip of the rete ridges (Watt 1998; Jensen etal., 1999). It is important to note that in the epidermis only afew basal cells express the differentiated-type K1/K10 keratins(Reigner et al., 1986). In contrast, the entire basal layer of thecentral cornea expresses K3/K12 keratins (Schermer et al.,1986; Chaloin-Dufau et al., 1990). Indeed, the corneal stem

5487Development 127, 5487-5495 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV2629

Recent work has shown remarkable plasticity betweenneural and hematopoeitic, as well as between hematopoeiticand muscle stem cells, depending on environmental stimuli(Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stemcells give rise to a proliferative transient amplifyingpopulation (TA), which is generally considered to beirreversibly committed. Corneal epithelium provides aparticularly useful system for studying the ability of TAcells to activate different genetic programs in response to achange in their fibroblast environment. Indeed, cornealstem and TA cells occupy different localities – stem cells atthe periphery, and TA cells more central (Lehrer, M. S.,Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci.111, 2867-2875) – and thus can be discretely dissected from eachother. It is well known that pluristratified epithelia ofcornea and skin display distinct programs ofdifferentiation: corneal keratinocytes express keratin pairK3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneousappendages, which express their own set of keratins. In our

experiments, central adult rabbit corneal epithelium wasthus associated either with a mouse embryonic dorsal,upper-lip or plantar dermis before grafting onto nude mice.Complementary experiments were performed using adultmouse corneal epithelium from the Rosa 26 strain. Theorigin of the differentiated structures were identified in thefirst case by Hoechst staining and in the second by thedetection of β-galactosidase activity. The results show thatadult central corneal cells are able to respond to specificinformation originating from embryonic dermis. They giverise first to a new basal stratum, which does not expressanymore corneal-type keratins, then to pilosebaceous units,or sweat glands, depending of the dermis, and finally toupper layers expressing epidermal-type keratins. Ourresults provide the first evidence that a distinct TA cellpopulation can be reprogrammed.

Key words: Cornea, Dermo-epidermal interactions, Differentiation,Epidermis, Hair follicle, Sebaceous gland, Sweat gland, Transientcells, Rabbit, Mouse

SUMMARY

Adult corneal epithelium basal cells possess the capacity to activate

epidermal, pilosebaceous and sweat gland genetic programs in response to

embryonic dermal stimuli

Corinne Ferraris 1,*, Genevieve Chevalier 1, Bertrand Favier 1, Colin A. B. Jahoda 2 and Danielle Dhouailly 1,‡

1Equipe Biologie de la Différenciation Epithéliale, UMR CNRS 5538, LEDAC, Institut Albert Bonniot, Université Joseph Fourier,Grenoble, France2Department of Biological Sciences, University of Durham, Durham, UK*Present address: L’Oréal Recherche, Centre Charles Zviak, 92583, Clichy Cedex, France‡Author for correspondence (e-mail: [email protected])

Accepted 29 September; published on WWW 14 November 2000

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and TA cells are not dispersed along the basal layer but occupydistinct localities in this layer. Studies in the rabbit suggestedthat the stem cells are localized at the periphery of cornea inthe limbus (Schermer et al., 1986; Cotsarelis et al., 1989), sincelimbal basal cells are slow cycling and lack expression ofdifferentiation markers K3/K12. More recent data obtained inmouse show that a hierarchy of TA cells are distributed fromthe periphery to the centrum of the corneal epithelium, whichconsequently contains only late TA cells (Lehrer et al., 1998).With regard to hair stem cells, they are located outside the hairmatrix: in the bulge, a portion of the upper mouse follicle thatis the follicular attachment site of the arrector pili muscle(Cotsarelis et al., 1991; Yang et al., 1993), or in the outer rootsheath in a region below the midpoint of the human hair follicle(Rochat et al., 1994). The concept of stem cells does notexclude the eventuality that there exists a hierarchy ofprogenitor cells that may revert back, may have differentialplasticity and may not be restricted to one tissue type.

Until recently, however, tissue-specific stem cells wereconsidered as unipotent, and their derived TA cells asirreversibly committed. New findings have radically alteredthis concept, at least in relation to stem cells. Thus, neural-derived stem cells produce a variety of blood cells when putinto irradiated mice (Bjornson et al., 1999). Likewise,hematopoeitic and muscle stem cells, both of mesodermalorigin, have remarkable plasticity depending on environmentalstimuli (for reviews, see Fuchs and Segre, 2000; Sealeand Rudniki, 2000). Moreover, the idea of adult cellreprogramming has gained general acceptance as a result ofcloning experiments in which it has been shown that adultnuclei can be reprogrammed to become totipotent when incontact with a zygote cytoplasm (Wilmut et al., 1997;Wakayama et al., 1998). The question thus arises as to whetheradult epithelial stem cell populations of the interfollicular skin,hair follicle and corneal epithelium are committed to theproduction of only one cell lineage – epidermal interfollicularstem cells giving rise only to the epidermis, follicular stemcells to hair follicle derivatives and corneal stem cells tocorneal epithelium (Miller et al., 1993). Alternatively, are theypluripotent and consequently of equivalent potentialities?In the second hypothesis, the epithelial stem cells maystill possess embryonic features and therefore theirmicroenvironment, the fibroblasts with which they areassociated, may play a crucial role in their differentiation.Studies of surgical wounds of different depths performed onpig flank show that re-epithelialization, which starts fromresidual appendageal structures, leads to an ordinary epidermiswhen originating from hair follicles, and to a thick plantar-likeepidermis when originating from the sweat glands (Miller etal., 1998). Thus, whereas all appendage stem cells are able togive rise to an epidermis, some differences may exist betweenhair and sweat gland stem cells. Likewise, when humankeratinocytes from different regions are transplanted onto nudemice, they retain their characteristics and appeared to becontrolled by intrinsic programs (Boukamp et al., 1990).

What would happen if the change in the fibroblastenvironment of the adult epithelial cells was more drastic, thatis if they were recombined with embryonic fibroblasts? Indeed,during embryogenesis, the epidermis is, in most cases,dependent upon its associated dermis for its differentiation.Heterotopic dermal-epidermal recombination experiments

between mouse plantar, upper-lip and dorsal embryonic skincomponents (Kollar 1970; Dhouailly 1977a,b) have shown thatthe choice between a glabrous or a hairy skin, between hairvibrissa and hair pelage-type follicles or between plantar anddorsal epidermis, is dictated by the origin of the dermis.Moreover, keratin analyses in recombinants involving postnatalplantar tissues (Delorme and Dhouailly, 1989) showed that theplantar-type keratin synthesis depends on the origin of thedermis. In the adult, the rat vibrissa follicle has long beenemployed as an experimental model. Its dermal papilla staysendowed with inductive hair properties of the embryonicdermal condensation, and, when associated with adultepidermis from different sources, even the plantar region(Reynolds and Jahoda, 1992; Jahoda et al., 1993) or theforeskin (Ferraris et al., 1997), is able to induce hair follicleformation.

In the latter experiments, the question remains as to whichepithelial cells of the adult epidermis can respond to newdermal influences, since the basal population contains bothstem and transient type cells dispersed throughout theepidermis? The adult cornea, however, has unique advantagesas an experimental system because stem and transient epithelialcells are regionally segregated. Here, we have investigated thecapacity of the epithelial component of adult mammaliancentral cornea, to follow an alternative differentiation pathwayby associating it with an embryonic dermis from a hair or asweat gland-forming region. As well as having a characteristickeratin expression, the cornea has two other advantages as asource of adult epithelium for cornea/skin recombination-typeexperiments – it is easy to manipulate, and it has noappendages. Contemporary investigations have underlined thenecessity of identifying the origin of responding cells whenexploring tissues interactions (Saha et al., 1989). Taking thiscaveat into account, and in order to identify unequivocally theorigin of the differentiated structures, bispecific epithelial-mesenchymal recombinants were performed, involving rabbitadult central corneal epithelium, and embryonic dorsal andplantar mouse dermis. Rabbit nuclei, which displayhomogeneous chromatin are easy to distinguish from mousenuclei which have bright spots of condensed chromatin whenHoechst stained (Cunha and Vanderslice, 1984). This systemcorresponds to the one that has been beautifully developed inbirds, between quail and chick (Le Douarin, 1973). Moreover,some experiments were performed using the Rosa 26 mousestrain, which allows cell origin to be distinguished by β-galactosidase staining (Zambrowicz et al., 1997). It should benoted that the rabbit corneal epithelium is thinner (Chaloin-Dufau et al., 1993), pliable and larger than the mouse one,allowing a larger number of recombination experiments to beperformed more easily.

We find that embryonic dermis causes the transformation ofadult central corneal epithelium, specifically to form anepidermis, and to differentiate either pilosebaceous units, whenconfronted with a trichogenic embryonic dermis, or sweatglands when confronted with a plantar dermis.

MATERIALS AND METHODS

AnimalsThe mice and rabbits were from Iffa-Credo and from Elevage

C. Ferraris and others

5489Epidermal potentialities of corneal basal cells

Scientifique des Dombes. The Rosa 26 mice (Zambrowicz et al.,1997) were from the Jackson laboratory. They were humanelyeuthanized. The nude mice onto which the chimaeric fragments weregrafted, were anaesthetized with an intraperitoneal injection of 100 µlof a solution containing 50 mg of valium and 1 mg of imalgene. Thiswas performed under French Animal Protection and Health Ministryauthorization number 04622 to D. D.

Isolation of hair- and sweat gland-inducing embryonicdermisEmbryonic trichogenic dermis was obtained from the upper-lip of12.5-day mouse embryos, and from the back of 14.5-day mouseembryo. Embryonic plantar dermis was dissected from the posteriorlimbs of 15.5-day mouse embryos. These were chosen becausethey were the optimal inducing stages in previous findings(Dhouailly, 1977a,b). The dermis was dissociated from itsassociated epidermis by enzymatic treatment 15 minutes (1.25%trypsin and 2% pancreatin), in Ca2+- and Mg2+-free Earle’s solution,then stored (10 minutes to 1 hour) in DMEM/20% foetal calf serumuntil use.

Preparation and grafting of the epithelial/mesenchymalheterospecific recombinantsOnly the central part of the adult rabbit cornea was used. This wascarefully excised using scissors, leaving a margin of around 5 mm atthe edges to avoid any contamination by limbal epithelium. The entiremouse corneal epithelium was excised, leaving just a margin of 1 mmnext to the limbus, because of its small size. The corneal epitheliumwas dissociated from its underlying stroma with 0.25% EDTA inCa2+- and Mg2+-free Earle’s solution, rinsed in Tyrode, and thenassociated with mouse embryo upper-lip, dorsal or plantar dermis onagar medium. After 1 hour these recombinants were grafted under thekidney capsule of athymic nude mice (Swiss nu/nu) according to amethod previously described (Higgins et al., 1989). The grafts wererecovered after 3, 6, 12 and 21 days, fixed in formalin for waxhistology, or embedded in OCT and stored at −80°C forcryosectioning.

Identification of the species origin of the cells in therecombinants To distinguish mouse and rabbit nuclei we used Hoechst staining. Thecompound 33258 Hoechst is a fluorochrome that binds to the narrowgroove of DNA and shows clear differences in chromatin patterningin the nuclei of rabbit and mouse (Ferraris et al., 1994, 1997). Thestaining procedure (modified from Cunha and Vanderslice, 1984)consisted of treating sections (wax or cryostat) for 15 minutes with a4 mg/ml solution of Hoechst 33858 in phosphate buffered salinesolution (PBS). Slides were mounted in 9:1 glycerol in PBScontaining sodium azide and 1.4 diazabicylo (2.2.2) octane (Dabco,Sigma), an anti-fading compound and were observed with amicroscope equipped with UV epifluorescence (wavelength 365 nm).In the case of recombinants involving tissues from the Rosa 26 mousestrain, the grafts were fixed in 2% paraformaldehyde, 0.2%glutaraldehyde in PBS, and then stained in 5 mM potassiumferricyanide, 5mM potassium ferrocyanide, 2 mM MgCl2, 50 mg/ml5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal; Sigma) inPBS, overnight at room temperature. Prior to histological analysis, thegrafts were postfixed in 4% paraformaldehyde for an additional 24hours, embedded in paraffin and sectioned. The slides werecounterstained with Hematoxylin. Some grafts were processed forroutine histology and stained with Hematoxylin.

Antibodies and immunofluorescent stainingIt is well established that the intermediate filaments of keratins areheterodimers composed of a basic and an acidic keratin (for a review,see O’Guin et al., 1987), thus in this study, we will use only twoantibodies, specific for the acidic member of each pair, epidermal or

corneal-type of keratins. The AK12 mouse monoclonal antibody,monospecific for the acidic corneal-type keratin K12 was produced inD. D.’s laboratory (Chaloin-Dufau et al., 1990), likewise the AK10 ratmonoclonal antibody for the acidic epidermal-type keratin K10(Delorme and Dhouailly, 1989). Cryosections (10 µm) were thawedand incubated according to standard protocols (Harlow and Lane,1988). For double staining, sections were incubated with theAK10, anti-rat FITC-conjugated antibody (Southern BiotechnologyAssociates, AL), and finally rhodamine-conjugated mousemonoclonal antibody AK12. After two final washes with PBS, slideswere immersed for 1 minute in a 5.10−7 M Hoechst 33258 solution(Sigma, Ref. B2883), washed twice in PBS and mounted in Mowiol4.88 anti-fading medium (Callbiochem, Ref. 475904).

RESULTS

Formation of a new basal stratum and of hair buds The central corneal epithelium of the adult rabbit is a fullydifferentiated non-keratinizing epithelium consisting of 6 to 7layers, a particular feature of which is the absence of a granularand of a cornified layer. Another difference with the epidermisis that in the central cornea, all the cells of the basal layerexpress the differentiating-type pair of keratins, as shownby K12 labelling in corneal epithelium immediately afterrecombination (Fig. 1A). At the dermal-epidermal junction,Hoechst labelling unequivocally delineated the mouse dermisand rabbit epithelium (Fig. 1A,B). After 3 days, inheterospecific recombinants of adult rabbit corneal epitheliumand embryonic mouse dermis from the plantar (15.5-dayembryo) or dorsal (14.5-day embryo) regions (n=5), a newbasal layer was formed, in which the cells no longer expressK12 at a high level (Fig. 1C). After 6 days, specimenscomprising 14.5-day dorsal dermis (n=7), all displayed earlyhair bulbs growing down into the dermis. Hoechst stainingclearly defined these early follicle stages as being derived fromthe corneal epithelium, and the dermal cells condensingunderneath (the dermal papilla precursors) as mouse-derivedcells (Fig. 1D). At this stage, in all specimens examined, theepithelium expressed K12 suprabasally (Fig. 1E).

Formation of a fully differentiated epidermis and ofhair folliclesRecombinants involving adult rabbit corneal epithelium and12.5-day upper-lip (n=17) or 14.5-day dorsal mouse dermis(n=27) were analyzed after 12 or 21 days. In every case hairfollicles formed. In specimens fixed after 21 days of grafting,in 100% of cases a huge amount of hairs were emerging. Insections, the corneal epithelium appeared to be transformedinto an epidermis characterized by a granular and a cornifiedlayer, associated with several pilosebaceous units (Fig. 2A).The rabbit origin of the epidermal cells and of the sebocytesand trichocytes, as well as the mouse origin of the dermal cells,were clearly identified by the Hoechst staining (Fig. 2B,C). Allthe nuclei in the epidermis, hair follicles and sebaceous glandsdisplayed an homogeneous staining (distinctive of rabbitepithelial corneal origin), while the hair follicle dermal papillaeand other dermal cell nuclei displayed intranuclear bodies(distinctive of mouse origin). Another way to distinguishbetween epidermal and dermal cell origin was to use mousetissues from the Rosa 26 strain. Relatively few successfulrecombinants were recovered, partly because the mouse

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corneal epithelium, which is stiffer than that of the rabbit, doesnot adhere well to the dermis, leading in more than 20 casesto only tiny patches of epithelial cells on the dermis.Nevertheless, in three cases (12 days after recombination), agroup of pilosebaceous units formed, in which the epithelialcells were clearly expressing β-galactosidase (Fig. 2D,E).

Formation of a polystratified epithelium and ofsweat glands Nineteen recombinants of adult rabbit corneal epithelium with15.5-day mouse embryonic plantar dermis were examined after21 days. Viewed externally, all the specimens showed theformation of footpads (Fig. 3A). Histology revealed sweatglands in the dermis and a thick epithelium with a cornifiedlayer (Fig. 3B). The precise stratification of the epitheliumvaried in different regions of the grafts. All grafts showedadditional epithelial cell strata, but not all grafts or all areashad a cornified layer – in some the morphology of the surfacelayers of the epithelium was quite cornea like (Fig. 3C). Thissuggested that the upper part of the epithelium in associationwith plantar dermis, remained longer with the corneaphenotype than recombinants with dorsal dermis, thisinterpretation being confirmed by the immunohistologicalstudy (see below). Detailed examination of the Hoechst stainedspecimens identified circular gland epithelium derived fromrabbit corneal cells, deep in the mouse-derived dermis (Fig.3D).

Different steps in the transition of adult cornealepithelium into an epidermisRecombinants of 12.5-day mouse upper lip dermis combinedwith adult rabbit corneal epithelium were examinedimmunohistologically after 12 and 21 days, with AK12 andAK10 antibodies. After 12 days, all specimens (n=6) hadproduced clearly recognisable hair follicles within amultilayered epithelium. Double labelling with K10 and K12antibodies revealed predominant expression of corneal type(K12) keratin suprabasally (Fig. 4A). It should be noted thatthe number of cell layers that express K12 is at least twice thatof a normal corneal rabbit epithelium. The basal layer of theepithelium and all cells of the hair follicles remainedunlabelled. Intriguingly, small patches of epidermal-type K10labelling were visible in restricted regions, just where thefollicles joined with the epithelium. Detailed examination ofthe hair follicles with Hoechst staining confirmed that thefollicle cells were rabbit in origin, while the dermal papilla andconnective tissue sheath were mouse derived (Fig. 4B). At 21days, fully formed pilosebaceous units are present in all of therecombinants of mouse upper lip dermis combined with rabbitcorneal epithelium (n=6). By this stage, staining with thecorneal-type (K12) antibody revealed only patchy labelling andin the higher and shedding layers of the epithelium (Fig.4C),while most of the lower epithelium stained with the epidermal-type K10 antibody. The labelling with the two keratinantibodies appeared to be strictly mutually exclusive. Thetransition between K12- and K10-synthesizing cells appearedto be delayed in the case of recombination with plantar dermis.Indeed at 21 days, (n= 3) a continuous layer of corneal-typeK12 marking was still present on top of a broader more basalK10-labelled layers (Fig. 4D).

DISCUSSION

In a series of experiments we show here that adult cornealepithelium, under the influence of embryonic skin dermis,undergoes a transition to an epidermal-type phenotype andproduces appendages (hair follicles or sweat glands)


Fig. 1.Formation of a new basal layer a few days after graftingrecombinants of adult rabbit corneal epithelium and 14.5-dayembryonic mouse dorsal dermis under the kidney capsule.(A) Initially the corneal epithelium (e) comprises 6-7 cell layers.Fluorescent labelling showing that all the corneal layers, includingthe basal layer, synthesize the K12 corneal-type keratin. (B) Sametype of specimen as A. The rabbit corneal epithelium (e) nuclei areuniformly stained, whereas the mouse dermal (d) nuclei exhibitbright fluorescent intranuclear bodies. The basal layer (bl) of theepithelium is composed entirely of rabbit cells. (C) As early as 3days after grafting, recombinants have formed a new basal layer(nwbl), which no longer expresses the K12 keratin. K12 labelling isonly present in the suprabasal layers. (D,E) 6 days after grafting.(D) The new rabbit basal layer has formed hair follicle buds (hb) inassociation with mouse dermal cell condensations, which willdevelop into the dermal papillae (dp). (E) The upper epithelial layersare stained with the AK12 antibody. (A,B,D) Hoechst staining; (B,D) paraffin sections; (A,C,E) cryostat sections fluorescent stainingwith AK12 antibody. Scale bars: 12 µm in A; 25 µm in B-E.


corresponding to the regional identity of the underlying dermis.In the dermal-epithelial recombination experiments presented,because the nuclei of cells from these two components belongto two different species, they are easily recognizable: Hoechststaining allows easy discrimination of mouse and rabbit cellnuclei. Likewise the X-gal staining of recombinants involvingthe Rosa mouse strain clearly ruled out contamination of theimplanted inductive dermal cells by epidermal cells of thesame origin.

While reports of reprogramming of adult (stem) cells arebecoming relatively prevalent (Bjornson et al., 1999), therehave been few observations of reprogramming of adult

epithelia. In one example, adult hair follicle dermal papillaewere shown to induce hair follicles when put in contact with avariety of epithelia (Oliver, 1970; Jahoda et al., 1993; Reynoldsand Jahoda, 1992; Ferraris et al., 1997). Others have used tissue

Fig. 2. Formation of hair follicles (pilosebaceousunits) induced in adult rabbit (A-C) or mouse (D,E) corneal epithelium by a mouse embryonichair-forming dermis, in kidney capsule grafts.(A) After 21 days, adult rabbit corneal epitheliumassociated with 14.5-day mouse embryo dorsaldermis. Longitudinal section showing a hair follicle(h) with associated sebaceous gland (sb). Note thatthe pluristratified epithelium now includes agranular layer (gl) and a cornified layer (cl), whichcharacterizes it as epidermal type. (B) Upper partof a pilosebaceous unit formed by a recombinantsimilar to A. Hoechst staining shows that the newlyformed epidermis (e) and sebaceous gland (sg) areentirely composed of rabbit epithelial cells withhomogeneous nuclei, while the dermis (d) containsonly mouse fibroblasts, displaying brightly spottednuclei. (C) Lower part of an oblique section of asimilar pilosebaceous unit as B, showing a hairmatrix (hm) consisting entirely of rabbit cells,associated with a dermal papilla (dp) formed bymouse cells. (D,E) Twelve days after grafting, arecombinant consisting of a mouse adult cornealepithelium from a Rosa 26 strain associated with a12.5-day mouse embryo upper lip dermis from awild-type strain. (D) The external view shows threeβ-galactosidase-positive hair follicles attached to apositively labelled Rosa epidermis. (E) Thelongitudinal section of the same explant shows twostained hair follicles (h) and their associatedsebaceous glands (sg), as well as the interfollicularepidermis (e). k, kidney. (A) paraffin section, Hematoxylin staining; (B,C) paraffin section, Hoechst staining; (D,E) galactosidase staining; (E)paraffin section counterstained with Hematoxylin. Scale bars: 50 µm in A; 25 µm in B,C; 1.5 mm in D; 450 µm in E.

Fig. 3. Formation of foot pads associated with sweat glands in adultrabbit corneal epithelium induced by 15.5-day mouse embryonicplantar dermis, 21 days after grafting under the kidney capsule.(A) External view of a recombinant showing six foot pads (fp).(B) Longitudinal section of a foot pad, showing an epidermischaracterized by an easily recognisable cornified layer (cl), and awell-formed sweat gland (sw). (C) Another example, showing apluristratified epithelium, which comprises more layers than acorneal epithelium but does not have either a granular or a cornifiedlayer. Its upper strata are morphologically similar to that of a cornealepithelium, with elongated nucleated cells (arrow). (D) Highermagnification of the lower part of an explant similar to A and B,showing the glomerular part of a sweat gland. The cells withhomogeneous nuclei lining the transversal sections of the gland (sw)originate from rabbit, while the surrounding dermis (d) is composedof mouse cells, distinguishable by their ‘dotted’ nuclei. (B,D) paraffin section, Hematoxylin staining; (C) paraffin section,Hoechst staining. Scale bars: 500 µm in A; 50µm in B; 25 µm inC,D.

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recombination work to study epithelial-mesenchymalinteractions in the reproductive system (Cunha, 1994). Thisgroup performed pioneering work (Cunha et al., 1980, 1983;Neubauer et al., 1983) which demonstrated mesenchyme-induced changes to epithelial gene expression. Recently, theyinvestigated whether epithelial differentiation could bereprogrammed in a fully differentiated adult human bladderepithelium (Aboseif et al., 1999) and found that neonatal ratseminal vesicle mesenchyme induced normal adult humanbladder urothelium to form glandular structures resemblingprostate.

All of the cornea recombination work done previously hasused embryonic corneas, and some class-specific differenceshave been reported. Thus corneal epithelium from the chickembryo is able to form scales or feathers when it is associatedwith either a tarsometatarsal or a dorsal dermis (Coulombreand Coulombre, 1971). However, in this species the cornealepithelium loses this capability after 7 days of incubation, andso may have limited plasticity (Zak and Linsenmayer, 1985).In contrast, corneal epithelium from rabbit embryos of all agesand even neonates can be induced to form hair follicles whenassociated with a dorsal embryonic dermis (Ferraris et al.,1994). This discrepancy is not surprising since a similarsituation is seen with skin epidermis from avian andmammalian species. Indeed, in chick embryos the epidermis ofthe mid-ventral apterium progressively loses its capacity toform feathers in response to dorsal feather-forming dermis(Sengel et al., 1969), whereas in mammals, even adult skinepidermis can form hair follicles in response to appropriatedermal influences (Oliver, 1970; Reynolds and Jahoda, 1992;Ferraris et al., 1997). It should be emphazised that the entirethickness of chick epidermis participates in the formation of

feather or scale buds, whereas only the basal epidermal layeris implicated in hair or sweat gland bud morphogenesis.

Our interspecific recombination experiments showed clearlythat signals from embryonic mouse dermis can be recognisedby, and elicit transformation of adult rabbit corneal epitheliumto epidermis, hair follicles and glands. Having said this, thedetailed chain of events, as revealed by the marking of stagesin the process, was also important as it provided greater insightinto questions relating to stem cell lineages and cellreprogramming. Most researchers working on epithelial stemcells, follow a fairly conventional model of stem cell activity,in which there is a progressive and irreversible transition fromstem cell to transient amplifying cells to differentiatedphenotype. Through a variety of different approaches, all workon mammals has suggested that for the cornea the putative stemcells reside in the limbal region. In rabbits, Schermer et al.(1986) and Chaloin-Dufau et al. (1990) showed thatmonoclonal antibodies specific to K3 or K12, respectively, twocorneal-type keratins, marked all cell layers of the cornealepithelium (in other words basal and suprabasal layers), butonly suprabasal layers of the limbus. In humans, Pellegrini etal. (1999) used cell culture and clonal analysis to investigateproliferative potential of epithelial cells from all over the ocularsurface and reported, similarly that corneal stem cells werelocated exclusively in the limbus. In mice, slow cycling,tritiated thymidine label-retaining cells were shown to besegregated in the limbal portion of the eye, while a hierarchyof TA cells occurs from next to the limbus to the centrum ofthe corneal epithelium (Cotsarelis et al., 1989; Lehrer et al.,1998). This relatively undifferentiated nature of basal cells inthe limbus, as well as their slow-cycling state are quoted asfurther evidence that stem cells reside there. In our experiments


Fig. 4.Formation of new suprabasal strata in adult rabbitcorneal epithelium associated with a mouse embryodermis, 12 (A,B) or 21 (C,D) days after grafting under thekidney capsule. (A-C) Recombinations with a 12.5-dayupper lip dermis. (A) After 12 days, the pluristratifiedepithelium (e) comprises numerous strata characterized bythe presence of corneal-type keratin K12 (red). Note thatthe basal layer (bl), as well as the forming hair follicles (h) are not labelled. A few cells localized at the point ofattachment between hair follicles and epithelium alreadysynthesize the epidermal-type keratin, K10 (green). (B) A higher magnification of the base of one of the hairshown in A, allows recognition of the bispecificity of theepithelial and dermal components. (C) After 21 days, asimilar recombination as A, shows that the cells containingkeratin K12 (red) are shedding, while continuous layers ofkeratin K10 (green) synthesizing cells has formed.(D) Recombination with a 15.5-day mouse plantar dermis.After 21 days there is still a continuous layer of corneal-type epithelium (K12) overlying newly formed epidermalstrata (K10). d, dermis; dp, dermal papilla. All specimensare cryosections with Hoechst staining and indirectimmunofluorescent staining with AK12 and AK10antibodies. Scale bars: 50 µm in A,C; 25 µm in B,D.


great care was taken to remove tissue from only the centralregion when dissecting the rabbit corneas. Thus from all theavailable evidence, the rabbit corneal epithelial cells used forour recombinations were a transient amplifying population.The observation that K12 was expressed throughout theepithelium at the time of grafting (time zero) was confirmatoryevidence that the corneal epithelium we used was centrallyderived and contained no stem cells.

Within a few days of being recombined with trichogenic orplantar mouse dermis, a new, non-K12 expressing basal layerwas produced by rabbit cells. In other words the epithelium hadformed a less-differentiated layer, taking on a phenotypeequivalent to the corneal limbus, or the basal layer of skinepidermis, both of which harbour stem cells. Thus it appearsthat the first stage of the transformation process may be therestoration of a more primitive or stem cell-like phenotypefrom amongst a TA cell population or even an alreadydifferentiating population. Recently, Lehrer et al. (1998)described modulation of the proliferative abilities of TA andstem cells in the cornea by injury or application of TPA. Theyshowed in particular that in response to wounding or chemicalstimulation, stem cells were mitotically stimulated, andtransient amplifying cells had an increased number of roundsof amplification. However, the change here is much morefundamental. Potten and Loeffler (1990) and Loffler and Potten(1997), re-examined many aspects of stem cell behaviour andput forward an elegant screw model to describe the process ofchange from stem cell to a mature, non-dividing phenotype. Aspart of this model, TA cells, that have left the stem-cell niche,are not irreversibly committed to a terminal differentiation (ormaturation) pathway, but are able to revert to being stem cellsin the event of stem cells being removed or destroyed. Ourexperimental observations appear to reflect these ideas veryclosely and may be supportive evidence for the model.

Our results are summarized in Fig. 5. They suggest that thefirst step in the transformation of the corneal epithelial basalcells to epidermis/follicular epithelium is their regression to amore primitive cell type. The observation that after a few days,the basal epithelial cells were both participating in hair folliclemorphogenesis and generating suprabasal K12-expressing

cells was also intriguing. This illustrates clearly thatproliferation and differentiation are not mutually exclusive.This is well documented by several studies in the field ofoncogenesis. For example epithelial-derived benign tumors canmaintain the same expression of keratins as the tissue of origin(Tsubura et al., 1991). This is another element of the screwmodel (Potten and Loeffler, 1990), which makes the point thatdifferentiation, i.e. expression of a new gene product, is notnecessarily linked to functional irreversibility. In ourexperiments it must be assumed that hair follicle stem cellswere also established during the process of folliclemorphogenesis. In this context it is significant that normal-looking full pilosebaceous units (hair with associatedsebaceous glands) were produced, since the appearance ofsebaceous glands (a subset of follicle epithelia) during folliclemorphogenesis must be indicative of stem cell attributes. In arecent paper, it was suggested that stem cells are localizedthroughout the epithelium of developing hair follicles in theembryo (Akiyama et al., 2000), suggesting that all hair budepithelial cells are potential stem cells, but that subsequentlythe stem-cell niche becomes progressively limited. Thedeveloping follicles induced in the adult corneal epithelium canbe considered as similar to embryonic follicles, which containlarge numbers of stem cells. There is clearly a close linkbetween hair follicle stem cells and epidermis, since outer rootsheath cells of the human hair follicle have been shown to beable to regenerate a fully differentiated epidermis in vitro(Limat and Noser, 1986; Lenoir et al., 1988). Likewise, afterskin injury, follicular cells migrate upwards to cover thewound, suggesting that they can serve as a source of skinepidermis (Lenoir et al., 1988). In relation to this, we showhere that the first signs of epidermal differentiation (K10expression) always appeared at the top of the developing hairfollicles. Therefore, this indicates that follicles may be theprimary source for epidermal homeostasis. This is inaccordance with a suggestion by the Barrandon group,according to which hair follicles are the main repositories ofepidermal stem cells (Kobayashi et al., 1993; Rochat et al.,1994), a view supported by Taylor et al. (2000).

The fact that adult corneal epithelium formed glandular

Fig. 5. Summary model of the effects of anembryonic hair-forming dermis on an associated adultcorneal epithelium. (A) Initially, all the corneal layerssynthesize the K12 corneal-type keratin (red). (B,C) A few days later. Formation of a new basallayer and of hair placode by the corneal cells, as wellas of new K12-expressing cell layers. (D,E) Two andthree weeks later. Differentiation of hair follicle andformation of new cell layers, expressing K10epidermal-type keratin (green). Note that the K10-expressing cells first appear at the point of connectionbetween the top of the hair follicle and the epithelium(D).

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epidermis in response to footpad dermis showed that thecorneal-epidermal transformation was a general phenomenonin such recombination-type experiments, and that ourexperiments agreed with a vast body of interaction work, inwhich the origin of the embryonic dermis determines theregional morphological characteristics of the skin and itsappendages (Kollar, 1970; Dhouailly, 1977a,b). Interestinglythe formation of epidermis in the plantar recombinations wasslower. This could be due to the fact that at the time (15.5 days)the embryonic plantar dermis is removed for the experiments,it is much less developed that embryonic dorsal skin dermis(14.5 days). Alternatively it could be that the large number ofinduced hair follicles provides a larger number of potentialepidermal stem cells, in comparison with the limited numberof induced sweat glands. Indeed, glands, like hair follicles arethought to be a source of epidermal stem cells (Miller et al.,1998).

Concerning the change in keratin synthesis, we show here aswitch from K12 (corneal-type) to K10 (epidermal-type in thedifferent skin regions, including plantar skin) keratin synthesis,which was induced by the embryonic dermis and is inaccordance with previous findings. However, we were unableto show the formation of an additional K9 keratin (plantar-typekeratin) by the rabbit corneal epithelium associated withplantar dermis (data not shown), perhaps due to the fact thatthis species, which does not have a distinct footpad, may nothave a functional K9 gene. In previous mouse/mouserecombination experiments, adult plantar dermis was shown tobe able to induce in an embryonic dorsal dermis not only theformation of foot pads, but also the synthesis of K9. In thereverse association, embryonic dorsal dermis combined withadult plantar epidermis not only led to the formation of hairs,but turned off the K9 gene (Delorme and Dhouailly, 1989).Likewise, although adult sole-derived keratinocytes continuedto express, or even re-expressed K9 when grafted on to musclefascia (Compton et al., 1998), they did not express it when co-cultured with non-palmar fibroblasts (Yamaguchi et al., 1999).In reverse, the same authors showed that non-palmarkeratinocytes co-cultured with palmar fibroblasts expressed K9keratin.

In conclusion, our results show that even in the adult, cornealepithelial cells retain the ability to transform into an epidermisand to produce hair follicles with associated sebaceous glandsor foot pads associated with sweat glands when recombinedwith embryonic mouse hair-forming or plantar dermis,respectively. Moreover we show that the formation of the newpluristratified epidermis originates from the induced hairfollicles, which confirms their role as the main repository ofepidermal stem cells (Rochat et al., 1994; Taylor et al., 2000).Thus, despite conditions that may modulate theirdifferentiation state in vivo, corneal epithelium and epidermalkeratinocytes have not diverged irreversibly duringembryogenesis and share common properties, reminiscent oftheir common embryological origin. Our results provide a firstclear indication that a distinct transient amplifying cellpopulation can be reprogrammed, and they imply that they doso by first reverting to a stem-like condition.

We are indebted to Dr Bruno Bernard, Dr Michel Demarchez, DrReiner Schmidt and Dr Hans Schaefer for their continuous interestand support. We thank the two reviewers for their helpful suggestions

and are indebted to Mrs Brigitte Peyrusse for the illustrations. Thiswork was supported by a fellowship from L’Oréal Research France toC. F., by a grant from CNRS and Université Joseph Fourier, and byan ARC grant number 5269 to D. D. C. A. B. J. was supported by theBBSRC.

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adult corneal epithelium basal cells possess the capacity...

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