Distinct contribution of stem and progenitor cells to epidermal maintenance

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1 ARTICLE doi:1.138/nture11393 Distint ontriution of stem nd progenitor ells to epiderml mintenne Guilhem Msré 1, Sophie Dekonink 1, Benjmin Drogt 1, Khlil Kss Youssef 1, Sylvin Brohée 1,2, Pngiot A. Sotiropoulou 1, Benjmin D. Simons 3,4 &Cédri Blnpin 1, The skin interfolliulr epidermis (IFE) is the first rrier ginst the externl environment nd its mintenne is ritil for survivl. Two seemingly opposite theories hve een proposed to explin IFE homeostsis. One posits tht IFE is mintined y long-lived slow-yling stem ells tht give rise to trnsit-mplifying ell progeny, wheres the other suggests tht homeostsis is hieved y single ommitted progenitor popultion tht lnes stohsti fte. Here we proe the ellulr heterogeneity within the IFE using two different induile Cre reominse oestrogen reeptor onstruts trgeting IFE progenitors in mie. Quntittive nlysis of lonl fte dt nd prolifertion dynmis demonstrte the existene of two distint prolifertive ell omprtments rrnged in hierrhy involving slow-yling stem ells nd ommitted progenitor ells. After wounding, only stem ells ontriute sustntilly to the repir nd long-term regenertion of the tissue, wheres ommitted progenitor ells mke limited ontriution. Skin epidermis is omprised of sl lyer of prolifertive ells nd severl suprsl lyers of terminlly differentited ells tht re progressively enuleted, forming squmes tht re shed from the skin surfe 1. Depending on ntomil lotion nd speies, the minimum trnsit time from the sl lyer to the ornified lyer hs een estimted t round 1 2 weeks 2. In homeostsis, ells lost during the ourse of turnover must e perfetly ompensted y ells generted in the sl lyer. Different theories hve een proposed to explin how this lne is hieved 1. On the sis of morphologil nd prolifertion studies, it hs een proposed tht the IFE is orgnized into disrete epiderml prolifertive units (EPUs), omprised of slow-yling stem ells (SCs) together with round 1 trnsit-mplifying ell progeny, whih undergo terminl differentition fter fixed numer of ell divisions 2. Following the lonl mrking of IFE ells y retroviruses 6 9 or mutgens 1,11, long-lived olumns of lelled IFE ells tht spn the epidermis from the sl lyer to the top of the ornified lyer pper, lending support to the onept of EPUs. By ontrst, the quntittive nlysis of linege tring dt in IFE using uiquitous promoter suggests tht tissue is mintined y single, equipotent, ommitted progenitor (CP) ell popultion in whih the lne etween prolifertion nd differentition follows from seemingly rndom ell fte deisions 12,13. Although this model is ttrtive in its simpliity, it does not esily explin the ility of tissue to respond rpidly to inresed ell demnd, suh s during wound heling, nd nnot rule out the presene of quiesent or slow-yling IFE popultion 14. More importntly, wheres the ontriution of different popultions of hir follile SCs to wound heling is well-estlished 1 18, little is known out the ontriution of puttive IFE SCs or CP ells to the repir of skin epidermis. From quntittive nlysis of linege tring dt using two different Cre reominse oestrogen reeptor (Cre-ER) trnsgeni mie, we demonstrte the existene of two distint popultions of epiderml progenitors tht form SC/CP ell hierrhy tht differentilly ontriute to the homoeostsis nd repir of the epidermis. Heterogeneity of epiderml progenitors To proe the prolifertive heterogeneity within the IFE, we mde use of two Cre-ER trnsgeni mie tht trget IFE progenitors (Supplementry Fig. 1, ). With high dose of tmoxifen, Cre-ER under the ontrol of the kertin 14 promoter (K14-Cre-ER) n trget most ells in the sl lyer of til IFE 19. A very low dose of tmoxifen (.2 mg) to K14-Cre-ER/RosYFP indued yellow fluoresent protein (YFP) expression t lonl density (Supplementry Fig. 1). Although Cre-ER under the ontrol of the involurin promoter (Inv-Cre-ER) predominntly trgets ells in the suprsl lyers, we hve reently shown tht some sl ells, hrterized y K ut not Inv protein expression, re lso indued ut t lower frequeny thn K14-Cre- ER (Supplementry Fig. 1d f) 2. The sl ells mrked y these two different Cre-ER omprise IFE progenitors, s demonstrted y their ility to proliferte, nd give rise to differentited progeny, forming olumns of lelled ells (Supplementry Fig. 1, d). To determine whether the two indution protools mrk the sme homogenous pool of progenitors, we first quntified their survivl t different time points fter tmoxifen dministrtion. Although mny of the lones indued y the K14-Cre-ER survived up to 1 yer postindution, most of the Inv-Cre-ER trgeted lones were progressively lost (Fig. 1 ). Wheres the popultion of surviving lones, defined s lones tht retin t lest one sl lyer ell, drops y round 3% for K14 mie from 4 weeks to 6 months fter lelling, the lone density flls y ftor of 1 in the Inv mie over the sme period, inditing tht IFE is mintined y heterogeneous pool of progenitors with different survivl potentil. Inv-Cre-ER trgets ommitted progenitors To gin further insight into the ehviour of these pprently distint pools of progenitors, we quntified the pttern of growth of individul lones trgeted y the Inv-Cre-ER t eight time points rnging from 3. dys to 48 weeks post-indution, period spnning more thn hlf of the verge lifetime of the mouse. At 3. dys, 4% of surviving lones hd undergone t lest one round of ell division (Fig. 2, nd 1 Université Lire de Bruxelles, IRIBHM, Brussels B-17, Belgium. 2 Université Lire de Bruxelles, Mhine Lerning Group, Brussels B-1, Belgium. 3 Cvendish Lortory, Deprtment of Physis, J. J. Thomson Avenue, Cmridge CB3 HE, UK. 4 The Wellome Trust/Cner Reserh UK Gurdon Institute, University of Cmridge, Tennis Court Rod, Cmridge CB2 1QN, UK. WELBIO, Université Lire de Bruxelles, Brussels B-17, Belgium. 212 Mmilln Pulishers Limited. All rights reserved MONTH 212 VOL NATURE 1

2 RESEARCH ARTICLE K14-Cre-ER/RosYFP K YFP 1 week 48 weeks Time post-lelling (weeks) d e Time post-lelling (weeks) f Frequeny (%) No. of totl ells No. of sl ells Inv-Cre-ER/RosYFP Surviving lone frtion (%) K14 Inv Time post-lelling (weeks) 1 week 48 weeks Figure 1 K14-Cre-ER nd Inv-Cre-ER trget IFE progenitors with different survivl hrteristis.,, Immunostining of K nd YFP in K14- Cre-ER/RosYFP () nd Inv-Cre-ER/RosYFP () til epidermis t 1 week nd 48 weeks post-indution showing the high survivl rte of K14-Cre-ER lones. Dshed lines represent the sl lmin. Hoehst nuler stining is represented in lue., Quntifition of surviving lones in K14-Cre-ER/RosYFP nd Inv- Cre-ER/RosYFP til epidermis t different times following tmoxifen dministrtion (n 327 lones t lest, for eh time point of K14 nd Inv). Symols show experimentl dt nd the lines represent the predition of the mthemtil modelling. The dt hs een normlized to the initil lone density s inferred y the fit to theory. Error rs show s.d. Sle rs, 2 mm. Supplementry Fig. 2), nd the vst mjority of these divisions resulted in one sl nd one suprsl ell (Fig. 2, d). At 4 weeks post-indution, lthough lmost ll surviving lones hd undergone t lest one ell division, 4% of lones still ontined only one sl ell (Fig. 2, e), inditing tht most of these divisions led to symmetri fte outome. However, the other 6% of surviving lones hd lso expnded slly (Fig. 2, f), inditing tht progenitors n lso symmetrilly self-renew. This lonl heterogeneity inresed progressively over the 48-week time ourse, inditing tht progenitors follow divergent ftes (Supplementry Fig. 2, ). At the sme time, the verge size of surviving lones (s mesured oth y their totl size nd their footprint on the sl lyer) grew pproximtely linerly with time (Supplementry Fig. 2d), wheres the numer of surviving lones progressively fell (Fig. 1). Suh ehviour mirrors tht reported previously 12 in studies using uiquitous promoter, nd indites tht Inv-Cre-ER trgets the tively yling CP ell popultion, in whih stohsti ell loss through differentition is perfetly ompensted y duplition (Fig. 2g). This onlusion is reinfored y quntittive nlysis of the dt, whih onfirms tht the sl lyer lone size distriution onverges onto the hllmrk sling ehviour in whih the hne of finding lone lrger thn some multiple of the verge YFP β4 g Committed progenitor Differentited ell 8% 1% 1% Division time : 1 per week 3. dys h Clone frtion (%) 1. w 1 w 2 w w 12 w 24 w 4 weeks stys onstnt over time (Supplementry Fig. 2e nd Supplementry Methods, setion Theory ) 21. From the detiled study of the sl nd totl lone size distriution over the 48 week time ourse (Fig. 2h, i, Supplementry Fig. 2f nd Supplementry Methods, setion Theory ), we re le to onlude tht, following CP ell division (t n verge rte of per week), 8% result in symmetri fte (leding to one dividing nd one differentited ell) with the reminder leding to symmetri duplition or differentition with pproximtely equl proility (Fig. 2g), onsistent with the results from ref. 12. Indeed, from the 48 week time point, we re le to resolve smll (less thn 1 in 14) is towrds terminl division. K14-Cre-ER trgets long-lived SCs To determine whether the dynmis of K14 lones re different from the Inv lones, we performed extensive lonl nlysis using the K14- Cre-ER mie rnging from 3. dys to 48 weeks post-indution (Fig. 3). At just 3. dys, 6% of K14 lones hd lredy undergone one round of ell division (Fig. 3, nd Supplementry Fig. 3), wheres the frtion of lones involving two or more sl ells ws gretly inresed ompred with Inv (Fig. 3 d). However, following n initil rupt expnsion, the growth of K14 lones rpidly deelertes over the first few weeks, with the size distriution from 8 weeks to 6 months post-indution showing only modest expnsion (Fig. 3 f nd Supplementry Fig. 3 ). Alongside the propensity of lones to persist, these oservtions re onsistent with the K14 promoter trgeting oth the CP ell popultion nd seond, slowyling, SC popultion. In prtiulr, the preipitous expnsion of lones (Fig. 3 ), whih is fr more rpid thn tht found in the 4 w 48 w i Clone frtion (%) w 1 w 2 w 8 w 12 w 24 w 4 weeks 4 w 48 w Figure 2 Inv-Cre-ER trgets IFE CP ells.,, Distriution of Inv-Cre-ER lone sizes s mesured y sl () nd totl () ell ontent of surviving lones, imged y onfol mirosopy on whole-mount til epidermis from 3. dys to 48 weeks following tmoxifen dministrtion. The numer of nlysed lones is indited in nd this is identil in, h nd i., Size distriution of surviving lones with more thn one ell t 3. dys postlelling disggregted ording to sl nd totl ell numer (n 37 lones). d f, Confol nlysis of representtive Inv-Cre-ER-trgeted sl lones t 3. dys (d) nd 4 weeks (e, f) post tmoxifen. Dshed lines represent the sl lmin. Hoehst nuler stining is represented in lue. g, A fit of the Inv-Cre-ER lonl fte dt predits tht progenitors undergo popultion symmetry through omintion of symmetri ell division, symmetril self-renewl nd symmetril differentition with n verge ell yle time of pproximtely 1 week. h, i, Frequeny distriution of sl (h) nd totl (i) lone size. Symols represent experimentl dt nd the lines orrespond to the model predition. Error rs show s.e.m. Sle rs, 1 mm. 2 NATURE VOL MONTH Mmilln Pulishers Limited. All rights reserved

3 ARTICLE RESEARCH YFP β4 g Time post-lelling (weeks) j Counts l H2B GFP d Stem ell Committed progenitor 3. dys 8% 1% 1% Division time : 4 6 per yer Unhsed h Clone frtion (%) Time post-lelling (weeks). w 1 w 2 w 1 8 w 12 w 24 w No. of totl ells No. of sl ells Inv study (Fig. 2 ), indites tht SCs re indued predominntly on ell yle entry. This onlusion is onsistent with the oservtion tht, fter dministrtion of 12-O-tetrdenoyl phorol-13-ette (TPA), 4 w 48 w H2B GFP Unhsed (no DOX) DOX: 6 weeks * e DOX: 9 dys * * 4 weeks DOX: 21 dys * Frequeny (%) i Clone frtion (%) k Frequeny (%) m YFP f w 1 w 2 w 4 w 8 w 8 w 12 w 24 w 2 48 w K14-Cre-ER 48 weeks 3 d 6 d 9 d 12 d 1 d 21 d Rounds of divisions Figure 3 K14-Cre-ER trgets IFE SCs.,, Distriution of K14-Cre-ER lone sizes s mesured y sl () nd totl () ell ontent of surviving lones, imged y onfol mirosopy on whole-mount til epidermis from 3. dys to 48 weeks following tmoxifen dministrtion. The numer of nlysed lones is indited in nd this is identil in, h nd i., Size distriution of surviving lones with more thn one ell t 3. dys post-lelling disggregted ording to sl nd totl ell numer (n 197 lones). d f, Confol nlysis of representtive K14-Cre-ER trgeted sl lones t 3. dys (d), 4 weeks (e) nd 48 weeks (f) post tmoxifen. Dshed lines represent the sl lmin. Hoehst nuler stining is represented in lue. Sle rs, 1 mm. g, Biophysil modelling of the lonl fte dt predits tht K14-Cre-ER trgets oth CP ells nd SCs with the ltter undergoing popultion symmetry through omintion of symmetri ell division, symmetril self-renewl nd symmetri differentition with n verge ell yle rte of etween 4 nd 6 per yer. h, i, Frequeny distriution of sl (h) nd totl (i) lone size. Symols represent dt nd the lines orrespond to the model predition. j, FACS nlysis of KtTA/tet(O)-H2BGFP mie t different time point following DOX dministrtion. k, Frequeny of the numer of ell divisions tht surviving sl IFE ells undergo during the hse period s quntified y FACS nlysis t different time points fter DOX dministrtion to dult KtTA/tet(O)- H2BGFP mie. Symols represent the verge of the experimentl dt (n 3 mie t eh time point). Lines orrespond to the predited model dynmis (Supplementry Methods, setion Theory ). l, m, Confol nlysis of H2B GFP immunostining in unhsed mie (no DOX) (left pnel) nd 6 weeks fter DOX dministrtion (right pnel), showing the preferentil loliztion of the slow-yling IFE ells in the ottom of epiderml undultions (strs) (l) nd of YFP immunostining in K14-Cre-ER/RosYFP 8 weeks fter tmoxifen dministrtion (m). Dshed lines re hir folliles. Sle rs, 2 mm. Error rs show s.e.m. Hoehst nuler stining is represented in lue. drug tht stimultes epiderml prolifertion, there is n 8% inrese in lone lelling fter low dose of tmoxifen (Supplementry Fig. 4, ). Furthermore, the deelertion in the rte of lonl expnsion t susequent times indites tht, fter division, SCs re-enter quiesent phse wheres their CP ell progeny go on to proliferte nd differentite. For these lones, the quiesent SC mother provides n nhor to the sl lyer (Fig. 3 f nd Supplementry Fig. 3), leding to lonl persistene. Suh ehviour is onsistent with the oservtion tht, in K14 mie, the level of positive regultors of the ell yle, suh s Cn1 nd Cd2, deresed, wheres negtive regultors of the ell yle, suh s Cdkn1 nd Cdkn2, inresed in sl ells etween 3. dys nd 8 weeks (Supplementry Fig. 4 e). With this hypothesis, if we ssume tht SC division n lso led to ll three possile ftes (Fig. 3g), the predited model dynmis provides n exellent fit to the lone size dt (sl nd suprsl) over the wide rnge of time points up to 1-yer post-lelling (Fig. 3h, i, Supplementry Fig. 3, d nd Supplementry Methods, setion Theory ). In prtiulr, we find tht SCs ount for some 6 % of the indued prolifertive ells. With SC division rte s low s 4 6 per yer, ftor of 1 2 times slower thn CP ells, most of the lone size dt (from 1 week to 3 months post-lelling) re lrgely fixed y the known hrteristis of CP ells. Drwing on the erly (3. dy) time point, we re le to dedue tht, in ommon with their progenitor ell progeny, SCs undergo popultion symmetri selfrenewl with some 8 6 1% of SC divisions resulting in symmetri fte (one SC nd one CP ell), wheres the remining divisions re eqully lned etween SC duplition nd symmetril differentition into two CPs (Fig. 3g). Finlly, lthough the nlysis points t prolifertive hierrhy, we nnot rule out the possiility tht prolifertive ells my swith reversily etween CP nd SC ehviour if the trnsfer rte is suffiiently low. Moreover, the dt do not llow us to infer whether the lne etween SC prolifertion nd differentition follows from intrinsi (ell utonomous) regultion or is ontrolled y environmentl ues (Supplementry Methods, setion Theory ) 21. Prolifertion dynmis of the IFE To hllenge these findings, we pplied further independent experimentl ssys to explore the IFE ell prolifertion using omintion of ell yle nlysis, -romo-2-deoxyuridine (BrdU) inorportion, nd pulse-hse experiments using the KtTA/tet(O)-H2B-GFP reporter mouse, tht hve proved useful in resolving quntittively the prolifertion dynmis of hir follile ulge SCs 1, We first determined the ell yle profile of sl IFE ells y determining their DNA ontent. These experiments show tht round 9% of 6 1 CD34 2 IFE ells were in the S/G2/M phse of the ell yle (Supplementry Fig., ). Continuous BrdU dministrtion lelled % nd 7 6 1% of K-expressing ells fter 24 nd 72 h, respetively, inditing n verge ell division time of round dys (Supplementry Fig., d nd Supplementry Methods, setion Theory ), onsistent with the inferred CP ell division rte. To develop more refined method to quntify prolifertion kinetis over longer period, we used KtTA/tet(O)-H2B-GFP mie (Supplementry Fig. 6). In the sene of doxyyline (DOX), histone 2B domin green fluoresent protein (H2B GFP) fusion protein is expressed t very high level in disrete pek of fluoresene (Supplementry Fig. 6, top pnels). Following DOX dministrtion, H2B GFP fluoresene dereses y ftor of two fter ell division 1,22 (Supplementry Fig. 6, ottom pnels). The disrete pek of H2B GFP fluoresene oserved y fluoresene-tivted ell sorting (FACS) nlysis during the hse period llowed n urte quntifition of the distriution in the numer of rounds of division experiened y sl lyer ells (6 1 CD34 2 ) t vrious time points following indution (Fig. 3j nd Supplementry Fig. 6d). At 3 weeks post-dox dministrtion, dilution of H2B GFP lel shows evidene of CP ell prolifertion, while smll pek t high levels of fluoresene is onsistent with round % or less of lel-retining ells (LRCs). 212 Mmilln Pulishers Limited. All rights reserved MONTH 212 VOL NATURE 3

4 RESEARCH ARTICLE From the quntifition of fluoresene over the time ourse, the distriution of ell divisions ws shown to e quntittively onsistent with the inferred SC nd CP ell dynmis (Fig. 3k nd Supplementry Methods, setion Theory ). Notly, fter 6 weeks of hse, the H2B GFP lel hs eome diluted in the vst mjority of sl lyer ells nd only smll minority retin high levels of expression (Supplementry Fig. 6d). Intriguingly, these LRCs re lolized to the ottom of the undultions of the til epidermis (Fig. 3l), whih orrelte with the lotion of persistent lones derived y the K14 ssy (Fig. 3m), nd re reminisent of the so-lled rete ridges in humn epidermis, where epiderml SCs were thought to reside 2,26. Moleulr hrteriztion of SCs nd CP ells To gin further insight into the moleulr properties of these two funtionlly distint pools of epiderml progenitors, we trnsriptionlly profiled FACS-purified 6 1 CD34 2 K14-Cre-ER nd Inv-Cre-ER YFP-positive IFE ells 3. dys fter tmoxifen dministrtion (Supplementry Fig. 7). Comprison of the K14 nd Inv trnsriptome reveled notle differenes. All mrkers previously ssoited with murine nd humn IFE SCs 2,27 29 were upregulted in K14-trgeted ells, inluding 6, 2, 3, 1 integrins nd Cspg4 (Supplementry Fig. 8). Rel-time quntittive reverse trnsriptse- PCR (qrt PCR) performed on independent iologil smples onfirmed the upregultion of these integrins in K14-trgeted ells (Fig. 4). FACS nlysis showed tht 64 integrins were expressed t higher nd more uniform level in K14- thn in Inv-trgeted ells. The pttern of 21 integrin showed more notle differene etween the two popultions, with very high nd nrrow pek of 21 expression in K14-trgeted ells ompred to the muh lower nd heterogeneous 21 expression in Inv-trgeted ells (Fig. 4, nd Supplementry Fig. 9). Gene ontology lssifition of the genes differentilly expressed y more thn 1.-fold etween K14- nd Inv-lelled ells reveled tht genes preferentilly expressed in mrna expression (fold over Inv) Fluoresene (% of sl epidermis) Itg6 Totl sl IFE K14 YFP + Inv YFP + Integrins Cell yle Cell signlling Chromosome segregtion 16 Totl sl IFE K14 YFP+ Inv YFP+ α2 β6 β1 Itg1 Itg2 Itg3 Cnd2 Cd β4 d mrna expression (fold over K14) Cd2 Cn1 Il1r Epgn Dll1 Nrp1 Kif11 Cenpp Totl suprsl IFE K14 YFP + Inv YFP + Elovl7 Cd Elovl6 F2h Degs1 Cenpe Kif14 Lpl Lss4 Cd36 Fds6 Dgt2 Olh Agpt3 Lipid metolism Counts Totl sl IFE K14 YFP + Inv YFP α2 integrin Elovl4 S13 Noth3 Grhl3 Ds1 Epiderml differentition Figure 4 Moleulr signture of K14 SC nd Inv CP ells., qrt PCR nlysis of the expression of representtive genes upregulted in K14-Cre-ERtrgeted ells, ompred with the level of expression of the sme genes in Inv-Cre- ER-trgeted ells nd totl sl IFE ells (n 3).,, Fluoresene histogrm () nd geometri men fluoresene () of integrin ell-surfe expression in ll sl IFE ells, K14-Cre-ER- nd Inv-Cre-ER-trgeted IFE ells, s determined y FACS nlysis, 3. dys fter tmoxifen (n 3). d, qrt PCR nlysis of the expression of representtive genes upregulted in Inv-Cre-ER-trgeted ells, ompred to the level ofexpression of the sme genes insl epiderml K14-Cre- ER-trgeted ells nd totl suprsl IFE ells (n 3).Errorrsshows.e.m. K14-positive ells re enrihed with very high sttistil signifine in funtionl groups involved in positive nd negtive ell yle regultion (for exmple, Cnd2, Cd2, Cdkn1, Cdkn2), mitosis (for exmple, Cn1, Kif11), hromosome segregtion (for exmple, Cenpe), regultion of ell prolifertion (for exmple, Epgn), nd DNA repir (for exmple, Br1) (Fig. 4, Supplementry Figs 8, 1 nd Supplementry Tle 1). By ontrst, Inv-Cre-ER lelled ells preferentilly expressed genes known to ontrol epiderml differentition 3 32 (for exmple, Noth3, Grhl3), kertinoyte differentition nd kertiniztion (for exmple, Sppr1, 1, 2d, 2i) 33, nd lipid metolism (for exmple, Elovl4, 6, 7, Olh), s onfirmed y qrt PCR nlysis (Fig. 4d, Supplementry Figs 8, 1 nd Supplementry Tle 1). Altogether these dt demonstrte tht K14- nd Invtrgeted ells differ y their gene expression profile, nd unover mny new mrkers preferentilly expressed y IFE SC nd CP ells. To hrterize further the moleulr heterogeneity of sl IFE ells, we FACS-purified different popultions of sl epiderml ells (6 1 CD34 2 ) ording to the level of 1 integrin expression whih, together with 2 integrin, present the most signifint differene etween K14 nd Inv-trgeted ells t 3. dys following tmoxifen dministrtion (Fig. 4 nd Supplementry Fig. 9). We frtionted epidermis into three distint popultions: one popultion (,3%) expressing very high levels of 1 integrin (whih re likely to e enrihed in SCs s they re only deteted in K14- ut not Inv-trgeted ells), seond popultion expressing intermedite levels of 1 integrin deteted in oth K14 nd Inv-trgeted ells (whih re likely to represent CP ells), nd ells expressing low levels of 1 integrin only found in Inv-trgeted ells (whih re likely to represent the differentited sl ells nd erly suprsl ells) (Supplementry Fig. 11). Strikingly, mny mrkers identified s eing upregulted in K14 versus Inv were lso upregulted in ells expressing very high levels of 1 integrin ompred to ells expressing intermedite or low levels (Supplementry Fig. 11). Similrly, mny genes preferentilly expressed y Inv ells were lredy upregulted in ells expressing intermedite levels of 1 integrin ompred to 1 integrin high ells ut, nevertheless, muh less expressed thn in ells expressing low levels of 1 integrin (Supplementry Fig. 11). These dt demonstrte tht the expression of mny differentition-ssoited genes is lredy upregulted t the erliest stge of progenitor ommitment, reminisent of the linege priming reported in hemtopoieti stem ells 34,3. Long-term SC ontriution to wound heling In ddition to their role in mintenne, dult SCs re implited in the repir of dmged tissue following injury. Linege tring hs shown tht hir follile SCs re moilized to the wound re nd ontriute to the repir of dmged epidermis At the sme time, geneti mouse mutnts, tht present omplete sene of hir follile, hel inisionl wounds with slight dely of the reepithelilistion, demonstrting tht IFE ells re lso ple of tissue regenertion 36. However, in unpertured onditions, the ontriution of IFE SCs nd progenitors to wound heling is urrently unknown. To ddress the respetive ontriutions of K14-SCs nd Inv-CPs during tissue repir, we first mrked sl progenitors y titrting the dose of tmoxifen so s to lel Inv nd K14-Cre-ER epidermis t roughly the sme density ( lones per mm 2 nd lones per mm 2 in K14 nd Inv, respetively), then sujeted til epidermis to full thikness exisionl wound using punh iopsy, nd ssessed the ontriution of mrked ells to repir (Fig. ). Whole-mount nlysis showed mjor reruitment of K14-Cre-ER-lelled IFE ells to the wound re, with lones migrting from the periphery towrds the entre of the wound (with only 7.% of lones from hir follile), persisting longterm fter wounding ( lones per mm 2 fter 3 dys) (Fig., nd Supplementry Fig. 12), nd onsisting of very lrge lones with rod sl tthment nd huge numers of differentited ells (Fig. d). In shrp ontrst, few Inv-Cre-ER-trgeted ells were reruited to the wound re, nd these lones were, on verge, muh 4 NATURE VOL MONTH Mmilln Pulishers Limited. All rights reserved

5 ARTICLE RESEARCH K14-Cre-ER/RosYFP TAM Wound d 1 d 14 d 21 d 3 d demonstrtes the ritil role of IFE SCs for the long-term repir of the epidermis, s hs een proposed 16. Inv-Cre-ER/RosYFP 8 w 12 w Anlysis K14-Cre-ER Inv-Cre-ER 1 dys 3 dys 1 dys 3 dys YFP β4 YFP d K14 3 d Inv 3 d smller. At 3 dys post-injury, few Inv lones survived ( lones per mm 2 ) nd onsisted of either lones of ells eing shed with no sl tthment (Fig. e), or smll lones with one or few sl ells (Fig. f). These dt demonstrte tht K14-derived SC re ple of extensive tissue regenertion, wheres Inv CP ells hve only limited ontriution to wound heling. Disussion Our study demonstrtes the existene, hierrhil orgniztion nd the prolifertion dynmis of two distint types of progenitors tht effet different funtions during homeostsis nd repir of the IFE in dult mie. Quntittive modelling of lonl fte dt suggest tht oth the slow-yling SCs nd the more rpidly yling CP ells shre similr pttern of popultion symmetri self-renewl in whih the lne etween prolifertion nd differentition is hieved through stohsti fte hoie (Supplementry Fig. 13). These findings provide reonilition of seemingly ontrditory theories of IFE mintenne 4,12, nd explin the prolifertive heterogeneity previously reported in IFE,37,38. The existene of slow-yling SCs nd more rpidly yling progenitors is reminisent of the sitution enountered in other tissues inluding orne 39,hirfollile 4, lood 41,42,musle 43 nd rin 44. In ll of these tissues, these slow-yling SCs n swith rpidly nd reversily etween quiesene nd tivity following injury nd/or drug tretment 1,4. Indeed, this prtitioning of funtion progenitors to undertke routine mintenne nd quiesent SCs to effet repir my represent generi strtegy of tissue mintenne. The demonstrtion tht the long-term wound heling potentil is dominted y the IFE SC omprtment my suggest tht either SC nd CP ell popultions re not interhngele, in ontrst to progenitors in intestinl epithelium 46, or tht their reversion rte is suffiiently slow to render its effets negligile. The seemingly short-lived ontriution of Inv CP ells to the repir of the epidermis is reminisent of the fte of K1 ulge-derived SCs during wounding 16, nd e f Inv 3 d Figure Mssive nd sustined ontriution of K14 SC during wound heling., Sheme representing the experimentl strtegy used to lel sl progenitors nd ssess their ontriution during wound heling., Mirosopi nlysis of YFP immunostining performed on whole-mount of wounded til epidermis 4 weeks fter tmoxifen dministrtion to K14-Cre- ER/RosYFP nd Inv-Cre-ER/RosYFP mie nd nlysed t different times following wounding. Dshed lines represent the wounded re. Sle r, 2 mm., Confol nlysis of whole-mount immunostining of YFP nd 4 integrin 21 dys following wounding, showing the deprture of K14 YFP lelled from the IFE. The rrow indites the wound loliztion. Sle rs, 2 mm. d f, Confol nlysis of representtive lones from wounded til epidermis 3 dys post-wound, showing very ig lones ontining multiple sl ells in K14 trgeted ells (d), wheres the rre still visile Inv mrked lones re either ompletely dethed from the sl epidermis (e) or very smll ontining one or few sl ells (f). Dshed lines represent the sl lmin. Sle rs, 2 mm. Hoehst nuler stining is represented in lue. METHODS SUMMARY Clonl YFP expression in the til IFE ws performed y dministrting.2 mg nd 2. mg tmoxifen to K14-Cre-ER/RosYFP nd Inv-Cre-ER/RosYFP mie, respetively. Immunostinings were performed s desried 47. Quntifition of the proportion of surviving lones s well s the sl, suprsl nd totl lone size ws determined y ounting the numer of YFP 1 ells using whole-mount til epidermis, nlysed y onfol mirosopy. BrdU pulse nd H2B GFP lel retention ws performed s desried 1,43. The wound heling ssy ws performed y dministrting 1 mg nd 1 mg tmoxifen to K14-Cre-ER/RosYFP nd Inv- Cre-ER/RosYFP mie, respetively. Four weeks lter punh iopsies were performed in the til epidermis nd nlysed t different time points. Mthemtil modelling of the lonl fte dt ws performed s desried in Supplementry Methods, setion Theory. For further detils see Supplementry Methods. Full Methods nd ny ssoited referenes re ville in the online version of the pper. Reeived 13 Mrh; epted 3 July 212. Pulished online 2 Septemer Blnpin, C. & Fuhs, E. Epiderml homeostsis: lning t of stem ells in the skin. Nture Rev. Mol. Cell Biol. 1, (29). 2. Potten, C. S., Sffhill, R. & Mih, H. I. Mesurement of the trnsit time for ells through the epidermis nd strtum orneum of the mouse nd guine-pig. Cell Tissue Kinet. 2, (1987). 3. Potten, C. S. Cell replement in epidermis (kertopoiesis) vi disrete units of prolifertion. Int. Rev. Cytol. 69, (1981). 4. Potten, C. S., Wihmnn, H. E., Loeffler, M., Doek, K. & Mjor, D. Evidene for disrete ell kineti supopultions in mouse epidermis sed on mthemtil nlysis. Cell Tissue Kinet. 1, (1982).. Potten, C. S. & Loeffler, M. Epiderml ell prolifertion. I. Chnges with time in the proportion of isolted, pired nd lustered lelled ells in sheets of murine epidermis. Virhows Arh. B Cell Pthol. Inl. Mol. Pthol. 3, (1987). 6. Mkenzie, I. C. Retrovirl trnsdution of murine epiderml stem ells demonstrtes lonl units of epiderml struture. J. Invest. Dermtol. 19, (1997). 7. Kolodk, T. M., Grlik, J. A. & Tihmn, L. B. Evidene for kertinoyte stem ells in vitro: long term engrftment nd persistene of trnsgene expression from retrovirus-trnsdued kertinoytes. Pro. Ntl Ad. Si. USA 9, (1998). 8. Ghzizdeh, S. & Tihmn, L. B. Multiple lsses of stem ells in utneous epithelium: linege nlysis of dult mouse skin. EMBO J. 2, (21). 9. Ghzizdeh, S. & Tihmn, L. B. Orgniztion of stem ells nd their progeny in humn epidermis. J. Invest. Dermtol. 124, (2). 1. Ro, S. & Rnnl, B. A stop-egfp trnsgeni mouse to detet lonl ell lineges generted y muttion. EMBO Rep., (24). 11. Ro, S. & Rnnl, B. Evidene from the stop-egfp mouse supports nihe-shring model of epiderml prolifertive units. Exp. Dermtol. 14, (2). 12. Clyton, E. et l. A singletype ofprogenitor ell mintins normlepidermis. Nture 446, (27). 13. Doupé, D. P., Klein, A. M., Simons, B. D. & Jones, P. H. The ordered rhiteture of murine er epidermis is mintined y progenitor ells with rndom fte. Dev. Cell 18, (21). 14. Jones, P. & Simons, B. D. Epiderml homeostsis: do ommitted progenitors work while stem ells sleep? Nture Rev. Mol. Cell Biol. 9, (28). 1. Tumr, T. et l. Defining the epithelil stem ell nihe in skin. Siene 33, (24). 16. Ito, M. et l. Stem ells inthe hirfollile ulge ontriutetowound repirut not to homeostsis of the epidermis. Nture Med. 11, (2). 17. Levy, V., Lindon, C., Zheng, Y., Hrfe, B. D. & Morgn, B. A. Epiderml stem ells rise from the hir follile fter wounding. FASEB J. 21, (27). 18. Snippert, H. J. et l. Lgr6 mrks stem ells in the hir follile tht generte ll ell lineges of the skin. Siene 327, (21). 19. Vsioukhin, V., Degenstein, L., Wise, B. & Fuhs, E. The mgil touh: genome trgeting in epiderml stem ells indued y tmoxifen pplition to mouse skin. Pro. Ntl Ad. Si. USA 96, (1999). 2. Lpouge, G. et l. Identifying the ellulr origin of squmous skin tumors. Pro. Ntl Ad. Si. USA 18, (211). 21. Klein, A. M. & Simons, B. D. Universl ptterns of stem ell fte in yling dult tissues. Development 138, (211). 22. Wghmre, S. K. et l. Quntittive prolifertion dynmis nd rndom hromosomesegregtion of hir follile stem ells. EMBO J. 27, (28). 23. Zhng, Y. V., Cheong, J., Cipurin, N., MDermitt, D. J. & Tumr, T. Distint selfrenewl nd differentition phses in the nihe of infrequently dividing hir follile stem ells. Cell Stem Cell, (29). 24. Zhng, Y. V., White, B. S., Shllowy, D. I. & Tumr, T. Stem ell dynmis in mouse hir folliles: A story from ell division ounting nd single ell linege tring. Cell Cyle 9, (21). 212 Mmilln Pulishers Limited. All rights reserved MONTH 212 VOL NATURE

6 RESEARCH ARTICLE 2. Jones, P. H., Hrper, S. & Wtt, F. M. Stem ell ptterning nd fte in humn epidermis. Cell 8, (199). 26. Lvker, R. M. & Sun, T. T. Heterogeneity in epiderml sl kertinoytes: morphologil nd funtionl orreltions. Siene 21, (1982). 27. Jones, P. H. & Wtt, F. M. Seprtion of humn epiderml stem ells from trnsit mplifying ells on thesis of differenes in integrin funtion nd expression. Cell 73, (1993). 28. Legg, J., Jensen, U. B., Brod, S., Leigh, I. & Wtt, F. M. Roleofmelnom hondroitin sulphte proteoglyn in ptterning stem ells in humn interfolliulr epidermis. Development 13, (23). 29. Tni, H., Morris, R. J. & Kur, P. Enrihment for murine kertinoyte stem ells sed on ell surfe phenotype. Pro. Ntl Ad. Si. USA 97, (2). 3. Rngrjn, A. et l. Noth signling is diret determinnt of kertinoyte growth rrest nd entry into differentition. EMBO J. 2, (21). 31. Blnpin, C., Lowry, W. E., Psolli, H. A. & Fuhs, E. Cnonil noth signling funtions s ommitment swith in the epiderml linege. Genes Dev. 2, (26). 32. Ting, S. B. et l. AhomologofDrosophil griny hed is essentil for epiderml integrity in mie. Siene 38, (2). 33. Cndi, E., Shmidt, R. & Melino, G. The ornified envelope: model of ell deth in the skin. Nture Rev. Mol. Cell Biol. 6, (2). 34. Månsson, R. et l. Moleulr evidene for hierrhil trnsriptionl linege priming in fetl nd dult stem ells nd multipotent progenitors. Immunity 26, (27). 3. Pin, C. et l. Inferring rules of linege ommitment in hemtopoiesis. Nture Cell Biol. 14, (212). 36. Lngton, A. K., Herrik, S. E. & Hedon, D. J. An extended epiderml response hels utneous wounds in the sene of hir follile stem ell ontriution. J. Invest. Dermtol. 128, (28). 37. Loeffler, M., Potten, C. S. & Wihmnn, H. E. Epiderml ell prolifertion. II. A omprehensive mthemtil model of ell prolifertion nd migrtion in the sl lyer predits some unusul properties of epiderml stem ells. Virhows Arh. B Cell Pthol. Inl. Mol. Pthol. 3, (1987). 38. Morris, R. J., Fisher, S. M. & Slg, T. J. Evidene tht the entrlly nd peripherlly loted ells in the murine epiderml prolifertive unit re two distint ell popultions. J. Invest. Dermtol. 84, (198). 39. Cotsrelis, G., Cheng, S. Z., Dong, G., Sun, T. T. & Lvker, R. M. Existene of slowyling liml epithelil sl ells tht n e preferentilly stimulted to proliferte: implitions on epithelil stem ells. Cell 7, (1989). 4. Cotsrelis, G., Sun, T. T. & Lvker, R. M. Lel-retining ellsreside in the ulge re of piloseeous unit: implitions for folliulr stem ells, hir yle, nd skin rinogenesis. Cell 61, (199). 41. Wilson, A. et l. Hemtopoieti stem ells reversily swith from dormny to selfrenewl during homeostsis nd repir. Cell 13, (28). 42. Foudi, A. et l. Anlysis of histone 2B-GFP retention revels slowly yling hemtopoieti stem ells. Nture Biotehnol. 27, 84 9 (29). 43. Roheteu, P., Gyrud-Morel, B., Siegl-Chedenier, I., Blso, M. A. & Tjkhsh, S. A supopultion of dult skeletl musle stem ells retins ll templte DNA strnds fter ell division. Cell 148, (212). 44. Bonguidi, M. A. et l. In vivo lonl nlysis revels self-renewing nd multipotent dult neurl stem ell hrteristis. Cell 14, (211). 4. Essers, M. A. et l. IFN tivtes dormnt hemtopoieti stem ells in vivo. Nture 48, (29). 46. Tked, N. et l. Interonversion etween intestinl stem ell popultions in distint nihes. Siene 334, (211). 47. Youssef, K. K. et l. Identifition of the ell linege t the origin of sl ell rinom. Nture Cell Biol. 12, (21). Supplementry Informtion is ville in the online version of the pper. Aknowledgements We thnk F. Bollet-Quivogne nd J.-M. Vnderwinden for their help with onfol imging. C.B. nd P.A.S. re herheur qulifié, G.M. nd S.B. re supported y fellowship of the FRS/FNRS. B.D. is supported y TELEVIE. C.B. is n investigtor of WELBIO. This work ws supported y the FNRS, the progrm d exellene CIBLES of the Wlloni Region, reserh grnt from the Fondtion Contre le Cner, the ULB fondtion, the fond Gston Ithier, the Europen Reserh Counil (ERC) nd the EMBO Young Investigtor Progrm. Author Contriutions C.B., G.M., B.D., S.D., P.A.S. nd B.D.S. designed the experiments nd performed dt nlysis. G.M., S.D., B.D. nd K.K.Y. performed ll the experiments. S.B. performed ioinformti nlysis of the mirorry. C.B. nd B.D.S. wrote the mnusript. Author Informtion The dt disussed in this pulition hve een deposited in the NCBI Gene Expression Omnius nd re essile through GEO Series ession numer GSE Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of the pper. Correspondene nd requests for mterils should e ddressed to C.B. ([email protected]) or B.D.S. ([email protected]). 6 NATURE VOL MONTH Mmilln Pulishers Limited. All rights reserved

7 ARTICLE RESEARCH METHODS Mie. K14-Cre-ER 19 nd TRE-mCMV-H2B-GFP 1 trnsgeni mie were provided y E. Fuhs. K-tTA mie 48 were gift from A. Glik. RosYFP mie 49 were otined from Jkson Lortory. Involurin-CreERT2 were previously desried 2. Mouse olonies were mintined in ertified niml fility in ordne with Europen guidelines. These experiments were pproved y the lol ethil ommittee (CEBEA). Trgeting YFP expression. For linege tring experiment, K14-Cre-ER/ RosYFP nd Involurin-CRERT2/Ros-YFP mie were indued etween 2 nd 4 months with.2 mg nd 2. mg of tmoxifen (Sigm-Aldrih), respetively, y intrperitonel injetion. For ell sorting, K14-Cre-ER/RosYFP nd Inv-Cre-ER/RosYFP mie were indued with 1 mg nd 1 mg of tmoxifen, respetively, y intrperitonel injetion nd isolted 4 dys lter. Prolifertion experiments. For H2B GFP hse, K-Tet off /TRE-mCMV-H2B- GFP were treted one with 2 ml of doxyyline (2 mg ml 21 ) y intrperitonel injetion nd simultneously ontinully fed with doxyyline food (1 g kg 21 ) until niml euthnsi. For BrdU experiment, mie were injeted every 12 h with 2 ml of BrdU (1 mg ml 21 ) nd nlysed 24 h nd 72 h fter the first injetion. Wound experiments. After mie nesthesi (% xylzine 1% ketmine in PBS), irulr piees of epidermis were removed from K14-Cre-ER/Ros YFP nd Inv-Cre-ER/RosYFP til epidermis, 4 weeks fter tmoxifen indution, using 3 mm dimeter iopsy punh (Stiefel). Histology nd immunostining. Skin epidermis ws removed from til one nd pre-fixed overnight in 4% prformldehyde t 4 uc. Tissues were wshed three times in PBS for min nd inuted overnight in 3% surose in PBS t 4 uc. Tissues were then emedded in OCT nd kept t 28 uc. Setions of 6 mm were ut using CM3S Lei ryostt (Lei Myrosystems). Setions were inuted in loking uffer (1% BSA, % horse serum,.2% Triton in PBS) for 1 h t room temperture. Primry ntiodies were inuted overnight t 4 uc or 1 h t room temperture in the drk. Setions were rinsed three times in PBS nd inuted with pproprite seondry ntiodies diluted to 1:4 nd Hoeshst in loking uffer for 1 h t room temperture. Setions were gin wshed three times with PBS. The following primry ntiodies were used: nti-involurin (rit, 1:1,, Covne), nti-k (rit, 1:1,, Covne), nti-k1 (rit, 1:1,, Covne), nti-4 (rt, 1:2, BD Biosienes), nti-gfp (rit, 1:1,, Moleulr Proes), nti-gfp (hiken, 1:1,, Am). The following seondry ntiodies were used: nti-rit, nti-rt, nti-got, nti-hiken onjugted to Alex Fluor 488 (Moleulr Proes), to rhodmine Red-X (Jkson Immunoreserh) or to Cy (Jkson Immunoreserh). Nulei were stined in Hoehst solution (1:2,) nd slides were mounted in DAKO mounting medium supplemented with 2.% Do (Sigm). Epiderml whole-mount. Piees of skin til were inuted in EDTA (2 mm) on roking plte t 37 uc for 1 h. Epidermis ws seprted from the dermis s n intt sheet nd wshed 2 times with PBS. Piees of epidermis were pre-fixed in 4% prformldehyde overnight t 4 uc or 1 h t room temperture. Epidermis were rinsed 2 times with PBS for min nd onserved in PBS with.2% zide t 4 uc. Smll piees of epidermis were inuted in loking uffer (1% BSA, % horse serum,.8% Triton in PBS) for 3 h t room temperture on roking plte (1 r.p.m.). The smples were inuted in primry ntiodies overnight t room temperture on the roking plte. The primry ntiodies used were the following: nti-k (rit, 1:2, Covne), nti-4 (rt, 1:1, BD Biosienes), nti-gfp (rit, 1:2, Moleulr Proes), nti-gfp (hiken, 1:2, Am). Smples were then wshed 3 times in PBS with.2% tween for 1 h nd inuted in pproprite seondry ntiodies diluted 1:4 in loking uffer overnight t 4 uc on the roking plte. Piees of epidermis were then wshed with PBS 3 times for 1 h. For BrdU stining, smples were inuted in HCl 1 M t 37 uc for 2 min, wshed with PBS,2% tween, stined with nti-brdu (rt, 1:2, Am) in loking uffer nd with pproprite seondry ntiody fter severl wshes. Nulei were stined in Hoehst solution diluted 1:1, for 3 min nd mounted in DAKO mounting medium supplemented with 2.% Do (Sigm). TPA experiment. 12-O-tetrdenoyl-phorol-13-ette (TPA) from Sigm hs een diluted in etone (finl onentrtion 2 mg ml 21 ). K14-Cre-ER/ RosYFP mie were topilly treted with TPA on til epidermis during 11 dys, indued with tmoxifen the 8th dys. The 12th dy, treted mie were killed nd proessed to get whole-mounts of til epidermis. Mirosope imge quisition nd quntifition. All pitures of setion immunostining were quired using the Axio Imger M1 Mirosope, the AxioCmMR3 or MrC mer nd using the Axiovision softwre (Crl Zeiss). Aquisitions were performed t room temperture using 32 numeril perture (NA).4 nd 34 NA.7 EC Pln-Neoflur ojetives (Crl Zeiss). All onfol pitures on whole-mount were quired t room temperture using Zeiss LSM78 onfol mirosope fitted on n Axiovert M2 inverted mirosope equipped with 34 NA 1.2 C-Apohromt wter immersion ojetive (Crl Zeiss MiroImging GmH). Sequentil snnings of 1,24 3 1,24 pixels, z-stk.3 mm, were quired using the ZEN 21 softwre (Crl Zeiss). Numer of sl lones per interfolliulr region were quntified on frozen setion using Axio Imger M1 mirosope for eh time point nd represented s perentge of sl lone. Clone sizes were quntified on whole-mount til epidermis y quiring individul lones using LSM 78 onfol mirosope nd the numer of sl nd suprsl ells were ounted. Dissoition of epiderml ells. Skin epidermis (from CD1, KtTA/tet(o)- H2BGFP treted or not with DOX, K14-Cre-ER nd Inv-Cre-ER/RosYFP indued with tmoxifen for 3. dys or 8 weeks) ws removed from til one nd inuted overnight in HBSS (Gio).2% trypsin (Gio) t 4 uc. Epidermis ws seprted from the dermis nd inuted on roking plte (1 r.p.m.) t room temperture for min. Bsl ells were mehnilly seprted from the epidermis y flushing 1 times under the epidermis. Tissues were then ut in piees of 1 mm 2 with slpel nd trypsin ws neutrlized y dding DMEM medium (Gio) supplemented with 2% Chelex fetl lf serum (FCS). Smples were filtrted on 7- nd 4-mm filter (Flon). Cell lelling, flow ytometry nd ell sorting. Using onentrtion of 2 millions ells per ml, ells were inuted in 2% FCS/PBS with primry ntiodies for 3 min on ie, proteted from the light, with shking every 1 min. Immunostining for isolting K14 nd involurin ells ws performed using iotin-onjugted nti-cd34 (lone RAM34; BD Biosienes) nd phyoerythrinonjugted nti-6 integrin (lone GoH3; BD iosienes). Primry ntiodies were wshed with 2% FCS/PBS nd ells inuted for 3 min in llophyoyninonjugted streptvidin (BD Biosienes) seondry ntiodies, on ie, with shking every 1 min. Living K14 nd involurin expressing epiderml ells were gted y forwrd stter, side stter, negtive stining for Hoehst dye nd y following the YFP signl. Bsl ells from the interfolliulr epidermis were trgeted using CD34-negtive 6-integrin-positive gting. Immunostining for isolting sl 1 high, intermedite nd low ells ws performed using iotin-onjugted nti-cd34 reveled with phyoerythrin-cy7-onjugted streptvidin (BD Biosienes) nd nti-cd29 llophyoynin-onjugted (1 integrin, lone Hm1-1, ebiosienes). Single living, CD34-negtive, 1 low/medium/high ells were sorted. 1 low, medium nd high gtes were set up on the sis of its expression in K14-Cre-ERnd Inv-Cre-ER/RosYFP-trgeted sl IFE ells. Fluoresene-tivted ell sorting nlysis ws performed using FACSAri I t high pressure (7 p.s.i.) nd FACSDiv softwre (BD Biosienes). Sorted ells were hrvested diretly in the lysis uffer provided y the RNesy mirokit (QIAGEN) supplemented with 1 ml of et-merptoethnol for every 1 ml of lysis uffer. RNA extrtion ws performed on freshly sorted ells ording to the mnufturer s protool. The entire proedure ws repeted in t lest three iologilly independent smples. Other integrin ntiodies were used to determine their expression in the different ell popultions: nti-cd49 phyoerythrin-onjugted (2 integrin, lone Hm2, BD iosienes), nti-cd29 llophyoynin-onjugted, nd nti-cd14 rt (4 integrin, lone A, BD Biosienes) reveled with n nti-rt Rhodmine Red x-onjugted (donkey, JksonImmunoReserh). Totl suprsl nd totl sl IFE ells isoltion for qpcr ontrols. For totl suprsl IFE ontrol, skin epidermis ws removed from til one nd inuted 1 h in EDTA (2 mm) on roking plte t 37 uc. Epidermis ws seprted from the dermis nd sl ells were flushed out from the epidermis. The piee of epidermis ontining the suprsl ells ws then frozen in liquid nitrogen. RNA extrtion ws performed using RNesy mirokit (QIAGEN): the tissue ws ut in smll piees nd lysed in RLT uffer ontining -merptoethnol ording to the mnufturer s protool. For totl sl IFE qpcr ontrol, ells were otined y gting the whole 6 1 (low medium nd high) nd CD34 2 popultion in FACS sorting. RNA qulity. Extrted RNA qulity ws tested using pillry eletrophoresis (Agilent Bionlyzer, Agilent RNA 6 Nno Kit). Degrded RNA smples or presenting sign of degrdtion or ontining high proteins or slt ontmintion were disrded. Mirorry nlysis. Cohort of 1 Inv-Cre-ER/RosYFP nd K14-Cre-ER/ RosYFP mie were used to isolte t lest 1, sl IFE ells (6 1 CD34 2 YFP 1 ) per replite. Totl RNAs were isolted from these sorted ells nd were lelled nd hyridized on mouse genome rry. Mirorrys were performed in duplite for the untreted K14-Cre-ER/RosYFP nd tmoxifentreted K14-Cre-ER/RosYFP nd Inv-Cre-ER/RosYFP sorted ells. All the results were normlized using the frozen roust multirry nlysis (frma) normliztion using R-ioondutor pkge frma,1 with stndrd prmeters. Geneti signtures were otined y onsidering genes presenting fold hnge greter on smller thn 2 or 22, respetively. 212 Mmilln Pulishers Limited. All rights reserved

8 RESEARCH ARTICLE Reverse trnsription nd quntittive PCR. Cohort of 1 Inv-Cre-ER/ RosYFP nd K14-Cre-ER/RosYFP mie were used to isolte t lest 1, sl IFE ells (6 1 CD34 2 YFP 1 ) per replite. Eh RNA smple ws quntified using nnodrop spetrophotometer. RNA qulity nd quntity used for qrt PCR ws extly the sme s for the RNA used for Mirorry nlysis. Purified RNA (2 ng) ws used to synthesize the first-strnd DNA using Supersript II (Invitrogen) nd rndom hexmers (Rohe). Mok ws otined following the sme proedure without dding Supersript II. Quntittive PCR nlyses were performed with 1 ng of omplementry DNA s templte, using Brillint II Fst SYBR QPCR Mster Green mix (Strtgene) nd n Agilent Tehnologies Strtgene Mx3P rel-time PCR system. All primers were designed using the Assy Design Center (Rohe pplied siene, The list of primers re shown in the tle underneth. The linerity of eh pir of primer used hs een tested with the following dilution of DNA: 8 ng, 2 ng,. ng,.12 ng. Anlysis of results ws performed with Mxpro softwre (Strtgene). Delt delt CT were used to lulte the reltive expression of involurin smples to the K14 smples using the housekeeping gene TtBox. Primers. Gene symol, forwrd primer nd reverse primer (9 to 39) re: Agpt3, tggtt nd tgggtg;cn1, tgttttgtttt nd gggggggttt; Cnd2, gttgtgg nd tttgtt; Cd36, ttgttttgtggttgg nd ttgtgttttgttttgtt; Cd, tgttgttggg gtgg nd tggtggttggtgt; Cd2, tgggtgtg nd tgtgggt tggt; Cd, gtgtttgtggtggt nd gtttgt; Cenpe, ttttgttgggtgg nd gggtttgttttt; Cenpp, ggtgtgg nd gttttg; Degs1, ttttgtgggtt nd ttggtttt; Dgt2, gggtttggt nd tggtgggttgtgtgt; Dll1, gggttttggtt nd tggttggggt; Ds1, ggggttttg nd ttttgggtt ; Elovl4, ggtgggtttt nd ggggttgt; Elovl6, ggg t nd ggggtgtgtggtg; Elovl7, ttgtggg nd gtgtg tttggg; Epgn, ggttgggggtttgtg nd ttgttttgtgt; F2h, tggtggtggg gg nd gttggtgtt; Fds6, tttttgt nd ttgtggggg tg; Grhl3, gggtgtgtgttg nd tgtttttgtggg; Il1r2, ttgtgtttt nd ggggttgtttg; involurin, tgtttg nd tgtttg; Itg2, gggggggtttt nd tgtttgttttg; Itg3, ggttgtggttgggtg nd ggttggtgt; Itg6, ttgggtgttggtg gt nd ttttttttggg; Itg1, tgggttgggtttgt nd tgtgg g; Kif11, gggggggg nd gttggtttg; Kif14, ggttttgt tgtttttg nd tttggtttttg; Kif2, gggggtgg nd ttggt gtggggg; Lss4, gtgtttgttttg nd tggtt; Lpl, tggt gggtgtgtgg nd tgggttgttgggggt; Noth3, gtgggttgggtgt nd gg gggttgtt; Nrp1, gggtgtttgtgttg nd tgtggggt; Olh, ggggtttggtt nd ggtttttgggtttt; S13, ggggttggtgg nd ggtgggtt; T-ox, gtggtttttgtt nd tgg ttgtggtg. 48. Dimond, I., Owoli, T., Mro, M., Lm, C. & Glik, A. Conditionl gene expression in the epidermis of trnsgeni mie using the tetryline-regulted trnstivtors tta nd rta linked to the kertin promoter. J. Invest. Dermtol. 11, (2). 49. Srinivs, S. et l. Cre reporter strins produed y trgeted insertion of EYFP nd ECFP into the ROSA26 lous. BMC Dev. Biol. 1, 4 (21).. Gentlemn, R. C. et l. Bioondutor: open softwre development for omputtionl iology nd ioinformtis. Genome Biol., R8 (24). 1. MCll, M. N., Bolstd, B. M. & Irizrry, R. A. Frozen roust multirry nlysis (frma). Biosttistis 11, (21). 212 Mmilln Pulishers Limited. All rights reserved