Journal of Cell Science CM6Q UK 6BQ, EC1M ora fCl cec 2,5690–5701 125, Science Cell of Journal 2012 August 20 Accepted o:10.1242/jcs.108167 doi: opieafml f2 oyetds oto hmeettheir exert them of Most Of polypeptides. 2003). 22 of Grose, family which and a (FGFs), factors comprise (Werner growth fibroblast are cytokines importance particular and factors the the growth identify and process to healing importance healing. wound major impaired normal underlying of the the mechanisms is control for that it burden factors financial system, enormous care an health generates significant causes in and healing common and impaired morbidity Because patients, 2007). particularly chemotherapy al., diabetic or et (Menke steroids a in of anti-inflammatory population, with is formation treated elderly patients the This the in in ulcers. problem resulting healing nonhealing the impaired, Unfortunately, chronic, frequently 2009). al., is et Sen 1997; process Martin, 2007; 2008; al., al., et et Menke (Gurtner scar reduced elasticity a exhibits and and although strength appendages site, tensile epidermal body this all injured lacks conditions, the that of remains clotting, normal repair Under complete blood to remodelling. leads involves tissue formation finally, which complex tissue and, a granulation initiated, injury, re-epithelialization, Upon is inflammation, repaired. needs process efficiently skin the and healing in rapidly defect be every of the environment, formation to the the covers in against and role barrier essential humans its a in to Owing organ surface. largest body entire the comprises skin The Introduction general a in skin. resulting wounded signaling, in FGFR migration of keratinocyte absence and of the a regulators words: velocity identified in key Key migration we components as defect, adhesion FGFs reduced this identify focal Underlying a results edge. major adhesions. had These wound focal of deficiency. of the expression keratinocytes turnover migratory at the FGFR1/2-deficient and migration in formation keratinocyte that reduction inefficient impaired identified to demonstrated significant we from owing mechanism, assays resulted persistence underlying that directional the transwell severely As impaired re-epithelialization is growth and keratinocytes. in repair of in wound delay variety wounding 2 that a and a show Scratch by 1 we and controlled (FGFR) Here, is contraction receptors disorders. process factor wound healing repair growth cause impaired The fibroblast can skin. lacking activity the or mice of expression in integrity abnormal delayed the their of and maintenance cytokines, the and for factors essential is repair wound Efficient Summary work § this to equally ` contributed authors *These 6 5 4 3 2 1 of Meyer Michael regulators key are migration 2 keratinocyte and 1 receptors FGF 5690 ihr Grose Richard rsn drs:TeVva .SihDprmn fNuougr,TeUiest fTxsHat cec etra oso,MdclSho,Houston, School, Medical Houston, at Center Science Health Texas of University The Neurosurgery, of Department Smith L. Vivian The address: Present uhrfrcrepnec ( correspondence for Author ue ayUiest fLno,BrsCne nttt,BrsadTeLno colo eiieadDnity nttt fCne,London Cancer, of Institute Dentistry, and Medicine of School London The USA and 63110, Barts MO Institute, Louis, Cancer St Barts Medicine, London, of of School University University Mary Washington Queen Biology, Developmental of Department Germany Martinsried, Switzerland 82152 Zurich, 18, Universite 8093 Faculte Klopferspitz Zurich, 634, Am ETH U Biochemistry, Sciences, INSERM of Health Institute Molecular Planck of Max Institute Biology, of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h on earpoesi rhsrtdb ag ubrof number large a by orchestrated is process repair wound The eNc ohaAtpls 8Aeu ars,013Nc,France Nice, 06103 Valrose, Avenue 28 Sophia-Antipolis, Nice de ´ G,Fclahso,Mgain eeihlaiain Wound Re-epithelialization, Migration, adhesion, Focal FGF, 1, 6 ,An-ahrn Mu Anna-Katharina *, n aieWerner Sabine and [email protected] ´deMe dcn vned aoboe 60 ieCdx0,France 02, Cedex Nice 06107 Valombrose, de Avenue ´decine ) 1,§ ller ¨ 1, ,Jnxa Yang Jingxuan *, nvitro in eetrepesdo eaioye Be ta. 00 Zhang 2000; al., et (Beer In keratinocytes 2003). on another Werner, expressed FGFR1-IIIb, activate and and receptor FGF22 normal (Steiling and FGF10 in skin FGF1, expressed addition, 1994). wounded FGF1, are in by al., which FGF22, activated particularly et and is FGF10 dominant-negative (Werner keratinocytes FGF7, a by keratinocytes expressing expressed severe in mice FGFR2-IIIb the in mutant by process FGFR2-IIIb reflected this keratinocytes was in on delay This receptors FGF Ortega process. re-epithelialization. activating 1989; repair control ligands al., 1998), et wound al., (Broadley the et formation angiogenesis tissue wound to for granulation important contribute and particularly is family FGF2 FGF Whereas the of Ornitz 2009; ligand different Mohammadi, 2001). by Itoh, and cells and characterized (Beenken IIIc are stromal specificities and receptor IIIb whereas binding the each variants, Significantly, of variants. IIIb variants IIIc the the mainly exclusively produce predominantly even express keratinocytes, or including IIIc. FGFR1–3 and cells, IIIb of designated Epithelial variants, domain FGFR alternative immunoglobulin-like two third generates alternative the by in achieved splicing is of complexity Ornitz Further splicing kinases, 2009; 2001). Mohammadi, tyrosine Itoh, and and receptor (Beenken four FGFR1–4 of designated activation through functions 1, eadohr rvosydmntae htdfeetmembers different that demonstrated previously others and We ` ailMoik Daniel , n nwuddskin wounded in and FGFR 2 rncit.Ms motnl,alternative importantly, Most transcripts. ilsPonzio Gilles , 3,4 ai .Ornitz M. David , eerhArticle Research X700 USA 77030, TX 5 , Journal of Cell Science n h neligmcaim aentbe characterized. been determined, not be have to mechanisms underlying remain the re-epithelialization and their mutant. wound and FGFR2-IIIb receptor(s) with to FGF dominant-negative ligands specific heterodimers of a contributions form might by the Therefore, FGFR1-IIIb to inhibited ligands, ability be common also to its response Through in FGFR2-IIIb 2006). al., et td edtrie h osqecso h oso these for of and loss repair the wound cutaneous of migration for consequences keratinocyte this keratinocytes In the in homeostasis. determined receptors cutaneous we the and study of function revealed regeneration, barrier appendage cells findings in These FGFR2 epidermal by and 2010). FGFR1 mitogens of al., roles et important keratinocyte epidermal (Yang of by dermis production underlying secretion chemokines involved and and that cells loop cytokines paracrine pro-inflammatory through double of hyperproliferation a of keratinocyte chronic induction The and caused junctions. occludin tight of inflammation abnormal and consequence of claudins a formation different as concomitant of barrier epidermal expression the This reduced in of inflammation. defect a cutaneous they by and caused progressive was appendages but epidermal of mild, loss developed mutant complete double a The showed keratinocytes. mice in mice characterized receptors and both or generated one recently lacking we skin, the in ligands ncnrlmc,weeswud nK-1R iewr still were mice K5-R1/R2 in wounds whereas mice, was control contraction, 1C). in (Fig. wound genotypes both reflects of mice which This in diameter, similar 1B). (Fig. wound that (distance dermis) given re-epithelialization, the diameter noninjured impaired of wound the because obviously the of was at to borders tongues morphometric relation the epithelial in between by the edges between determined wound distance was both as the the closure at mice wound of area in measurement K5-R1/R2 delay small a in a However, detected only 1A). clot, (Fig. comprised a edge with tissue filled wound granulation were K5-R1/R2 the wounds the and genotypes, injury, control both and after in of mice 3 similar In was day mice. wounds At the S1B. of Fig. shown appearance schematically material 1B,C). are supplementary (Fig. wound the in mice of K5-R1/R2 components in different The healing but impaired the mild, confirmed defect. the 2010) healing al., that the severity et for in (Yang indicating responsible mice increase not these shown), is not in did not seen inflammation and in (data progressive age) observed aging of already weeks was upon (6 phenotype but mice The Fig. 1A–C). young slight, K5-R1/ (Fig. in material a impaired mice severely showed (supplementary R2 was healing mice healing wound However, K5-R2 in S1A). 2004). delay al., all nonsignificant in et FGFR1-IIIb to (Zhang lacking months mice cells with 5 results or previous 3 confirming 1.5, process. of healing the age affected the inflammation the at performed whether mice determine we 2010), with al., experiments et the mild full-thickness (Yang develop inflammation mice mutant progressive double generated mice) but the (K5-R1/R2 (K5-R1 that receptors FGFR1 Given both we keratinocytes. lacking or in mice mice) of (K5-R2 process, FGFR2 back mice), the healing on for wounds keratinocytes excisional in wound FGFR2 and FGFR1 the of role the determine To Results ourvltefnto fFF1II,FF2II n their and FGFR2-IIIb FGFR1-IIIb, of function the unravel To tdy5atrwudn,adnegauaintsu a formed had tissue granulation dense a wounding, after 5 day At histomorphometry subsequent with analysis Histological ages, all at healing wound normal showed mice K5-R1 nvitro in . GR nkrtnct irto n on ear5691 repair wound and migration keratinocyte in FGFRs nlmaoyclswsntovosyatrdi h FGFR granulomatous no the these was in of there altered distribution importantly, obviously and Most not mice. number was mutant an The (supplementary cells with Ly6G S1D). myofibroblast immunostaining inflammatory marker by Fig. neutrophil revealed of material the as against healing consequence antibody of stage the any mice in not K5-R1/R2 and of abnormal is abnormalities. the wounds myofibroblasts that indicating contraction the S1C), by Fig. in material (supplementary populated of location area the myofibroblast in tissue differences obvious of detect granulation not wounds against lack could 5-day we obvious antibody of However, sections and an stained mice we with wound 2007), the in (Hinz, myofibroblasts of of contraction further role important increased movements the K5- Given even in the contraction. diameter case to wound the not the owing diameter was where impaired this mice wound of However, demonstrating R1/R2 mice, contraction. result the control of in a 5 onset of day the part and 3 reduction in day between least Thus, occurred 1B, at (Fig. contraction. importantly, point was time wound Most This this 1A). at panel). (Fig. delayed middle strongly clot was a closure wound with filled predominantly onso 5R/2mc.Ti yohssi ute supported healing further the is in hypothesis This impaired results mice. is These K5-R1/R2 of panel). migration wounds right keratinocyte 2D, that (Fig. compensation suggest mice proliferation reflecting the K5-R1/R2 enhanced probably of in animals, by length reduced control the slightly to wounding, only after compared 5 was day epidermis because this At at wound tissue, repair. proliferate wound of epidermis stage wound the the early into in ability keratinocytes migrate the this few to reflects only At epidermis keratinocytes panel). wound left the the 2D, of of (Fig. length injury the after point, 3 time epithelium double- day wound in at the reduced mice of significantly was (Yang knockout area length similar mice its the mice, in K5-R1/R2 of K5-R1/R2 in spite of In observed dermis 2010). of al., already the expression et enhanced in was the mitogens as from increase keratinocyte resulted and wounds This skin nonwounded 5-day 2B,C). (Fig. of studies 5-bromo-2 the by epithelium within determined increased The wound even 2A). (Fig. was injury hyperproliferative proliferation after keratinocyte mild, 5 of a day only rate at showed and reduction wounding nonsignificant and after but control of 3 day in loss at similar the mice K5-R1/R2 was of the of epithelium consequence area wound direct the Interestingly, hyperproliferative a keratinocytes. in be FGFR2 to and FGFR1 likely was defect this at delay the for responsible stages. was later was contraction whereas delay wound re-epithelialization, the of repair impaired reduction of by phase caused early observed predominantly the 1A,C). animals, During the (Fig. mice. mutant was tissue K5-R1/R2 granulation double However, of the staining area extended green). in an larger had collagen S1E, they much and still of Fig. were wounds late material intensity the (supplementary similar of where staining, Goldner density Masson by a the revealed as mice genotypes and between both comparable of was panel), tissue scar right tissue/early granulation 1A,B, (Fig. healed contraction. impair could that reaction eas i o bev necsieifamtr epneat response inflammatory excessive an observe not did also We enx oue nteipie eeihlaiain because re-epithelialization, impaired the on focused next We in delayed significantly was healing wound together, Taken fully were genotypes all of mice wounding, after 14 day At 9 doyrdn Bd)incorporation (BrdU) -deoxyuridine a sot uceatn( actin muscle -smooth a -SMA). Journal of Cell Science oiidtaselasy rmr Fg A n immortalized K5-R1/R2 and from a S2A) 3A) Fig. In (Fig. material S4). (supplementary primary S2, keratinocytes assay, Figs material transwell supplementary modified 3A–C; (Fig. K5-R1/ with mice and control R2 experiments from migration keratinocytes immortalized performed lacking and primary we keratinocytes of FGFR2, capacity and migratory FGFR1 the study further To efficient for migration required the keratinocyte are have FGFR2 not and did FGFR1 arrows). tip by the indicated at elongated 2E, cells (Fig. with the appearance and flattened tongue mice thicker, K5-R1/R2 epithelial in generally tongue epithelial was flat from the front, a the keratinocytes at keratinocytes formed Whereas mice tongue: of control epidermal tip the migrating at keratinocytes the the of appearance morphological the by hair HF, epidermis; wound hyperproliferative HE, tissue; granulation G, eschar; Es, epidermis; E, dermis; D, clot; 500 Cl, bars: H/E. Scale mice. with K5-R1/R2 stained in were closure wounds wound Delayed 1. Fig. 5692 n on lsr B n on imtr(itnebtentebrest h oijrddri)()wr eemnd naeaeo w ons ahfr each mean wounds, represent two of Bars average point. An determined. time were per (C) analyzed dermis) noninjured were the genotype, to borders per the mice between (distance six diameter least wound and (B) closure wound and ora fCl cec 2 (23) 125 Science Cell of Journal m .Arw on otetpo h irtn oge ftewudeiems ( epidermis. wound the of tongues migrating the of tip the to point Arrows m. ( A aafnscin rmtemdl f3dyod(d) -a-l 5W n 4dyod(14dW) 14-day-old and (5dW) 5-day-old (3dW), 3-day-old of middle the from sections Paraffin ) 6 GRdfcetadcnrlkrtnctswssilosre nthe in observed still was keratinocytes between control difference and The FGFR-deficient 3B,C; S3). and (Fig. S2B assays Figs scratch material supplementary in factor. confirmed growth was exogenous keratinocytes R1/R2 an but of 2010), that absence cells the al., the in of et seen deficiency also (Yang migratory was of (EGF), intrinsic EGF responsiveness the reduced to reflects factor a rather FGFs cells of for because FGFR1/2-deficient growth substitute not the was partially This epidermal of only 3A). can presence (Fig. EGF the of that in mice demonstrating capacity concentrations K5-R1/R2 migratory reduced from high a cells defined showed Surprisingly, even in mice. but wild-type, K5-R1/R2 rate from FGF10 not keratinocytes and migration of FGF7 migration stimulated reduced of further Addition strongly medium. serum-free a keratinocyte showed mice s.e.m. h eeemgaoydfc fpiayadimraie K5- immortalized and primary of defect migratory severe The B , C /-tie on etoswr nlzdmorphometrically, analyzed were sections wound H/E-stained ) follicle. mat om Journal of Cell Science i ftemgaigtnu)wsdtrie tbt on de.A vrg ftowud rma es ee iewsaaye e iepitadgenotyp and mean point represent time A–D per analyzed in was Bars mice epidermis. arrows). seven t wound by least to hyperproliferative (indicated at edge from wound HE, mice wounds the K5-R1/R2 tissue; two from in of (distance tongue average epidermis An nonflattened wound edges. broad, the wound of both length at The determined morphometrically. was ( analyzed tongue) were migrating mice the K5-R1/R2 of and tip control of wounds 5-day-old and ta. 07,w nlzdteeprmtr nour in parameters (Hartwig these persistence analyzed the we directional (velocity), 2007), the al., speed and et migratory migration, the of on direction depends the that closure Given S2B). scratch Fig. collagen material on (supplementary as dishes well I-coated as 3B,C) (shown Fig. proliferation in keratinocytes cell immortalized inhibits for which C, mitomycin of presence ( genotype. and point time per analyzed was mice three least ti at per from analyzed wounds were two ( of mice wounds. average six 5-day-old An least for edges. at shown wound both are from at mice wounds determined K5-R1/R2 two was and of control average from An sections wounds. mice. Representative 5-day-old ( K5-R1/R2 and genotype. of 3-day-old and skin from point wounded sections in H/E-stained impaired using is morphometrically migration Keratinocyte 2. Fig. essec a eeeyipie,adte i o irt into migrate not did they and velocity impaired, directional severely reduced their was Furthermore, 4B). a persistence (Fig. with cells control migrated to 2). compared consistently 1, cells Movies K5-R1/R2 material (supplementary and keratinocytes control two from K5-R1/R2 using keratinocytes immortalized layer immortalized of cell migrating, lines the independent scratching of after taken recordings were keratinocytes Live-cell 4A–E). (Fig. E ersnaieHEsandscin rm3dyodwud hwn h i ftewudeiems oetefatndwudtnu ncnrlmice, control in tongue wound flattened the Note epidermis. wound the of tip the showing wounds 3-day-old from sections H/E-stained Representative ) B elpoieainwsaaye yBd noprto tdy5atrwudn.Tewudi ntergthn ieo h sections. the of side right-hand the on is wound The wounding. after 5 day at incorporation BrdU by analyzed was proliferation Cell ) nvitro in system GR nkrtnct irto n on ear5693 repair wound and migration keratinocyte in FGFRs h ulu fms el rmK-1R ie(i.4,right 4F, (Fig. mice asterisks). 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Journal of Cell Science oa dein F)wsaaye yimmunofluorescence paxillin phosphorylated by or total analyzed against was antibodies into of with (FA) formation staining seeding Concomitantly, adhesions after red). focal rhodamine- 5B, points with (Fig. time staining phalloidin by coupled different plates coated at I/fibronectin collagen mice mutant and on observed of from was reduction concentrations cells dishes. minor a I-coated to different Only collagen 5A). compared with (Fig. reduced mice coated control significantly dishes was PBS-coated on fibronectin on or with keratinocytes dishes assays FGFR1/R2-deficient adhesion of data, performed adhesion preliminary our we with Consistent finding, keratinocytes. substrate immortalized this cells. the control quantify to to compared adhesion To delayed was their spreading that subsequent noticed and from we keratinocytes mice immortalized K5-R1/R2 and primary of passaging Upon K5- of formation keratinocytes adhesion R1/R2 focal and attachment Impaired 5694 atclryovosa ae iepit fe cacigo the was of this scratching after and points mice, 4H). time (Fig. wild-type later with monolayer from at mice obvious cells K5-R1/R2 particularly from to cells compared observation, more this ruffles with significantly Consistent were 2005). there inefficient al., reflects et ruffles (Borm membrane migration of formation enhanced that enx nlzdteatnctseeo fclsfo control from cells of cytoskeleton actin the analyzed next We thspeiul ensonfrclue ua keratinocytes human cultured for shown been previously has It ora fCl cec 2 (23) 125 Science Cell of Journal rvd rbbeepaainfrtemgaoydeficiency. findings migratory these the FA FAs, for migration of explanation that and probable turnover spreading suggests a and provide formation that finding rapid Given unevenly This the impaired. require FAs. also were control large is of 10% formed turnover such than less formed contrast, had By cells 5B,C). that (Fig. had FAs large FAs very genotypes and compared both The cells distributed, K5-R1/R2 of controls. in FAs cells fewer with 5B. were seeding, Fig. there but in after attached, shown are minutes examples contrast, exhibited Representative Ninety By and FAs. rounded few periphery. still a were cell only mice K5-R1/R2 the cells from at cells control most distributed seeding, FAs uniformly and after spreading, were and minutes attachment efficient showed Twenty reproducibly green). 5B, (Fig. eaioye eeldn infcn ifrnei surface subunits in integrin major difference the of significant of levels expression no expression immortalized the revealed of analysis affects keratinocytes and cytometry FGFR1 Flow keratinocytes of integrins. 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FA mechanisms and the adhesion determined next We paxillin and keratinocytes kinase K5-R1/R2 adhesion in focal of expression Reduced oto essK-1R eaioye,adteewsas no also was there integrin and in keratinocytes, difference K5-R1/R2 versus control or n 4husatrwudn.Br ersn mean represent Bars wounding. after hours s.e.m. 24 and points hours time 4 the different ( at in wounding. taken incubated scratch were then after Pictures were C. cells mitomycin was the of wound and presence scratch monolayer, A the confluency. into to inserted grown Bars were side. mice upper K5-R1/R2 the and onto were cells membrane of mean the seeding represent of after side hours bottom 24 the the counted to at added Cells was modified (Ctrl.) medium. a vehicle culture or using EGF capacity FGF10, FGF7, migratory assay. their transwell for analyzed were mice i.3 oso GR n GR fet irto fcultured of migration affects FGFR2 and FGFR1 keratinocytes. of Loss 3. Fig. N § 12. , ( 5 fteclshdbtenoeadten and one between had cells the of 25% 6 A rmr eaioye rmcnrladK5-R1/R2 and control from keratinocytes Primary ) ...( s.e.m. b ciaina sesdb idn fthe of binding by assessed as activation 1 C B h radvi fclswsdetermined was cells of devoid area The ) .Imraie eaioye rmcontrol from keratinocytes Immortalized ). a v, a and 6 6 b 1in Journal of Cell Science cac sa xeietwr ae ocutclswt ufe.Fu iepit ftelv mgn xeietwr hsn h ecnaeo cells of mean percentage represent The chosen. Bars were points. experiment imaging time live indicated the the of points at time determined Four ruffles. was with cells cells count all to among taken were experiment assay scratch a narw xmlso oplrzdclsaeidctdwt natrs.Tesrthi ttetpo h itrs cl a:50 bar: Scale pictures. the of ind top are the cells at polarized of is Examples scratch mice. 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Fig. rmr eaioye sn etr ltig(supplementary blotting western S4C). Fig. using material and immortalized keratinocytes not for confirmed primary was (data mice the K5-R1/R2 levels of and control expression expression similar low The shown). indicating detectable, barely E C epniua oeet(c nA.Br ersn mean represent Bars A). in (‘c’ movement perpendicular ) ipaeet(ierdsac ewe trigadedpit b nA,( A), in ‘b’ point; end and starting between distance (linear displacement ) H ersnaiepcueo irtn elwt rmnn ufei hw ntergthn ie ufe r niae yawiedt itrsof Pictures dot. white a by indicated are Ruffles side. right-hand the on shown is ruffle prominent a with cell migrating a of picture representative A ) a 1, a 2, a ,and 5, A aaeesaaye o h uniiaino irto.Temgaigclswr nlzdfr( for analyzed were cells migrating The migration. of quantification the for analyzed Parameters ) b eeudtcal or undetectable were 3 a uui nclsfrom cells in subunit 6 6 ...( s.e.m. 6 ...Amnmmo i ifrn itrsfo w ifrn ellnsprgntp was genotype per lines cell different two from pictures different six of minimum A s.e.m. 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B velocity, ) og apparatus Golgi ihruffles with ctdwith icated elline cell zed Journal of Cell Science sn natbd gis the against antibody an using 5696 el ecudntdtc n eoie aii 3 ( 332 minutes) laminin (45 deposited any assay the detect adhesion of not detachment the could upon we for and cells S4B), used the Fig. we material at for (supplementary genotypes that staining both point weak of time mice a from only keratinocytes the immortalized revealed with cells correlate permeabilized S4A). not Fig. material did matrix (supplementary levels extracellular cells the this expression of of genotype the expression but the protein, in variability some opnns efcsdo ailnadF iae(FAK), kinase hepatocyte for FA required be and to shown paxillin FA were proteins major on these of because focused phosphorylation We and/or and result components. could expression FAs adhesion reduced of particular, turnover from possibly In impaired and formation keratinocytes. impaired the the FGFR1/R2-deficient be to of for likely migration are responsible mechanisms other predominantly Therefore, shown). not (data utemr,imnfursec nlsso tahdand attached of analysis immunofluorescence Furthermore, ora fCl cec 2 (23) 125 Science Cell of Journal c -hi flmnn32 hr was There laminin-332. of 2-chain c 2-chain) c 2in eosrt htFF otosterepeso nkeratinocytes. findings in These expression their 6C–E). controls (Fig. FGF7 within FGF7 that level of demonstrate protein addition in and after increase RNA the hours an at 12–24 found genes reproducibly these of we levels expression immortalized paxillin, the the and for When analyzed FAK and total extent. keratinocytes FGF7 of recombinant HaCaT similar with human treated or a were mice control to (Y- from reduced FAK keratinocytes phosphorylated were and 397) of (Y-118) levels paxillin proteins, paxillin a these and As of phosphorylated expression S4D). reduced FAK the for Fig. per of confirmed of material consequence was lines (supplementary FGFR2 levels and keratinocytes cell FGFR1 the primary of absence independent in the in three reduction proteins with The This confirmed genotype. 6B). (Fig. was FGFR2 and FGFR1 result FA of these absence the of in expression components reduced strongly immortalized a from revealed lysates al., protein keratinocytes et of (Ishibe analysis migration blot Western and 2004). spreading cell induced factor growth rd.Clswr nlzdb ofclmicroscopy. 50 confocal bars: by Scale analyzed were Cells (red). phalloidin rhodamine-conjugated was using cytoskeleton visualized actin The phosphorylated (green). or paxillin total against I- stained antibodies collagen were with on Cells minutes dishes. 90 fibronectin-coated or and/or 20 for were attach mice to allowed K5-R1/R2 and control from keratinocytes aeil n ehd.Tredifferent in Three described Methods. as and seeding Materials or after I, minutes collagen 45 BSA, fibronectin on adhesion were for mice analyzed K5-R1/R2 and control from keratinocytes ellns asrpeetmean 100 represent independent Bars with lines. reproduced cell was result 680 least The at cells. including line, cell per determined; photomicrographs was mice) cells K5-R1/R2 all from cell; among cells per on ten asterisks and by one indicated (between FAs large with cells ( control arrows. with in indicated FAs are shown. are genotypes different n and 2.5 (1, used 5 were fibronectin of concentrations i.5 mardahso n Afrainof formation FA keratinocytes. and K5-R1/R2 adhesion Impaired 5. 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FGFs suggesting only to EGF, FGF7 not al., including response did mitogens, in exogenous et mice migration impaired of (Ceccarelli K5-R1/R2 are show from keratinocytes effect and keratinocytes human Surprisingly, migration potent of keratinocyte the migration for with signaling consistent receptor FGF migration. impaired the the keratinocytes for of of proliferation compensated stages strong obviously later the At however, a process, migration. as repair keratinocyte delayed impaired was of re-epithelialization result wound proliferation keratinocyte mice, enhanced K5-R1/R2 al., even or in autonomous. et normal cell the not (Yang of is spite hyperproliferation In phenotype this show that demonstrating from not 2010), keratinocytes site did cultured wound mice inflammation, Furthermore, the K5-R1/R2 1994). cutaneous at al., et show proliferation in (Werner keratinocyte not mutant reduced FGFR2-IIIb did exhibited dominant-negative which finding, this a keratinocytes, with Consistent expressing 2010). was al., develops and mice et that hyperproliferation (Yang skin inflammation animals nonwounded progressive these slight the in in from seen result a already to shown was even latter The and observed. of skin mice, wounded rate in impaired K5-R1/R2 the not was Interestingly, proliferation keratinocyte migration. of and proliferation more keratinocyte the crucial of is particular re-epithelialization. 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Mohammadi, and A. Beenken, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108167/-/DC1 online available material Supplementary months. 12 after release Sciences. for Life number PMC Molecular in in Health program [grant Deposited graduate of were/are Zurich A-K.M. the Institutes Foundation and TH-08 of M.M. members National D.M.O.]. number Science to the HL105732-01 [grant number and [grant National Zurich S.W.]; to. ETH Swiss 310030_132884/1 by the supported 06-3]; was work This Funding kindly for Heidelberg, Petra keratinocytes. Center Dr HaCaT Research suggestions, providing helpful Cancer for Aumailley, German Germany, Boukamp, Monique floxed Cologne, Dr of the and University for Germany, Finland, Martinsried, Helsinki, Biochemistry, of mice, transgenic University fgfr1 Jose K5-Cre Partanen, the Dr Juha for ETH assistance, Dr Spain, Fernandes, technical Madrid, Andreia CIEMAT, excellent Jorcano, and for Switzerland Born-Berclaz Zurich, Christiane thank We examining Acknowledgements experiments for used was test * U groups. Mann–Whitney Software between Pad CA). differences (Graph Jolla, software PRISM La the Inc., using performed was analysis Statistical analysis Statistical mM 1 containing 7.4, pH PBS in Heidelberg, BSA 50 (Serva, 1% with I Ca using blocked were collagen performed wells or All substrates. was (Invitrogen), fibronectin Coating BSA, Germany). with (PBS) In noncoated coated either 2005). were al., that or plates et 96-well (Czuchra into seeded described were previously keratinocytes brief, as performed were assays Adhesion assay adhesion Cell plates transwell matrix-coated with performed were (8 assays migration Transwell assay migration Transwell a with microscope points M time 200 Zeiss different a at used and photographed we was scratching imaging area cell same after The 10 made live directly tip. was For pipette scratch contrast sterile thereafter. 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