ilg,TeUiest fIw,Iw iy A522 USA. 52242, IA City, Iowa Iowa, of University The Biology, USA. 52242, IA City, iy A522 USA. 52242, IA City, nefrnrgltr atr6rgltskrtnct migration Biggs C. keratinocyte Leah regulates 6 factor regulatory ARTICLE RESEARCH ß 2840 2014 April 10 Accepted 2013; July 31 Received in 2 humans, In 1 2006). (Ingraham al., anomalies et Richardson epidermal 2006; and as al., craniofacial lethality, et limb, perinatal exhibit as Irf6 during well essential lacking is and Irf6 Mice IRFs, (Honda most embryogenesis. infection to contrast viral In after 2006). Taniguchi, response interferon 2006). al., the et mediate Ingraham role 2011; crucial al., et a Botti playing 2012; (Irf6) al., et 6 (Biggs the regulatory with interferon factors, transcription factor by regulated tightly of keratinocytes is of differentiation and growth keratinocytes coordination The of 2003). differentiation (Coulombe, and proliferation the migration, the dependent ultimately on repair is Epithelialization requires 2005). dermal Arpey, and and (Baum healing angiogenesis epithelialization, inflammation, wound Cutaneous INTRODUCTION Migration, 6, Keratinocytes factor regulatory RhoA Interferon the WORDS: KEY through functions migration. Irf6 GTPase- cellular regulate that Rho to suggest Takenpathway a keratinocytes. data Irf6-deficient Arhgap29, these in together, of reduced was expression closing , in activating The Blocking delay the RhoA keratinocytes. wounds. rescued active RhoA, wild-type scratch of and effector in downstream fibers observed a stress ROCK, that of with network compared increased keratinocytes an Irf6-deficient exhibited Interestingly, process. during persisted scratch-healing that the phenotype a were molecular spread, keratinocytes more Irf6-deficient and However, larger underlying min. consistently 60 after the matrices similarly all adhere to understand keratinocytes investigated. Irf6-deficient and were wild-type To that adhesions show We cell–matrix Irf6. and cell–cell lacking mechanisms, speed cells reduced and migration in directional deficient of showed imaging Live scratch wound. the scratch a closing in delayed compared were keratinocytes wound embryos. their that close wild-type hypothesis to with failed the post-conception days tested 10.5 embryos Irf6-deficient at we adhesion. and study, migration cellular this regulates Irf6 In differentiation. and proliferation keratinocyte regulates (Irf6) 6 factor regulatory Interferon ABSTRACT Ato o orsodne([email protected]) correspondence USA. for 48824, *Author MI Lansing, Human East and University, Pediatrics State of Michigan and Development, Genetics Molecular and Microbiology ra .Schutte C. Brian nedsilnr rdaePormi eeis h nvriyo oa Iowa Iowa, USA. of 52242, University IA The City, Genetics, Iowa in Iowa, Program of Graduate University Interdisciplinary The Pediatrics, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,24–88doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. r6blnst h R aiyo rncito atr,which factors, transcription of family IRF the to belongs Irf6 4 3 1,2 eateto iceity h nvriyo oa Iowa Iowa, of University The Biochemistry, of Department eeomna tde yrdm ak eatetof Department Bank, Hybridoma Studies Developmental ahleL Naridze L. Rachelle , 5 n atn Dunnwald Martine and nvitro In r6dfcetmrn embryonic murine Irf6-deficient , 1 rsA DeMali A. Kris , 5 eatet of Departments 1,2, * 3 ailF Lusche F. Daniel , nspaaa eaioye fteeiemsadpasacrucial a plays and differentiation epidermis epidermal expressed the in is role Irf6 of Although keratinocytes healing. suprabasal wound consistent in in 2010), IRF6 for al., role et a (Jones with cleft non-syndromic than surgery with cleft to patients corrective likely (Kondo following more complications were disorders wound VWS with have clefting patients Interestingly, 2002). orofacial al., et two syndrome, pterygium IRF6 Katnne l,20) diinly eietfe Arhgap29, identified we Additionally, TGF 2002). directed al., mediating et for (Kaartinen TGF for activation of necessary RhoA formation the and 2011). for al., but required also et is (Jackson differentiation migration keratinocyte epidermal for al., et McMullan 2005; al., 2003). et (Grossi differentiation keratinocyte olwdb h iasml fodroe frarve,see review, a migration (for of ones coordination The older 2005). of contacts al., et cell–matrix disassembly Vicente-Manzanares and keratinocyte the cell–cell by of in followed assembly the function includes its that 2006), unknown. al., currently is et migration Ingraham 2012; h elcnrcinncsayfrtasoain(ilyadHall, and support (Ridley that translocation and for 1992). complexes necessary contraction adhesion stress cell at assembling the anchored for are responsible is that GTPase interest fibers small (GAPs, 2008; particular main Ridley, Of the 1997). and RhoA, D’Souza-Schorey, factors Heasman and activating 2011; exchange Aelst al., Van GTPase nucleotide et (Guilluy and guanine cycle inactivating) of activating) forms GTPases control (inactive) (GEFs, dynamics. GDP-bound the and cytoskeleton through (active) GTP-bound actin between of cells other regulators with and substrate 2000). the al., with et adhesions form (Vasioukhin the proteins of assembly these the for that the necessary turn, is in adhesions actin and, of environment, contraction linear the and about integrins information into requires for cytoskeleton cadherins evolve The 2000). subsequently al., et (Vasioukhin that with E- contacts allow cells Simultaneously, initial to adjacent form 2010). on adhesions disassemble Geiger, located cell–cell and cadherin-mediated integrins, and of assemble 2008; (Zaidel-Bar clusters Carlier, that migration include and protrusions, cues Clainche cellular (Le such and cell diverse, of are the cues sensors Migratory of 2005). of al., tail et disassembles Vicente-Manzanares event and the cell), cellular a retract the (two of to edge filopodia in leading and the but, lamellipodia defining protrusions of contents, cell formation cellular the and the the reorganizes drives Clainche scaffold to cellular Le this organization see migration, review, and dynamic provides a normally structure cytoskeleton (for actin The cytoskeleton reorganization 2008). the Carlier, by actin driven is the it achieve of to required force the and ellrmgaini ihycodntdbooia process biological coordinated highly a is migration Cellular ebr fteRofml fsalGPssaetecentral the are GTPases small of family Rho the of Members as a e od ydoe(W)adpopliteal and (VWS) syndrome Woude der Van cause nvivo In nvitro In oee,Ro a enfudt edispensable be to found been has RhoA however, , tde eosrt oefrRo Taein GTPase RhoA for role a demonstrate studies 4 pne Kuhl Spencer , b inln uigpalatogenesis during signaling 3 nvivo in 4 ai .Soll R. David , and b -nue tesfibers stress 3-induced nvitro in Bgse al., et (Biggs 4 ,

Journal of Cell Science euae h ci yokltni eaioye n alters and keratinocytes Irf6 that in hypothesize cytoskeleton we 2006), migration. actin al., cellular et the Xu TGF of 2006; regulates effector downstream al., a et is 1997). and (Knight candidate al., 2006) al., et al., cleft et (Ingraham et palatogenesis novel Saras Knight proper 2006; 2012; a for al., required as is et Irf6 RhoA, (Leslie Because Irf6 for of affinity downstream high with GEF a ARTICLE RESEARCH a e1. MCukyadMri,19) eas ftesevere the that of embryonic Well-established Because at 1995). described Martin, resection embryo. and (McCluskey hindlimb been (e)11.5 after day the have closure epidermal to models follow wound-healing animals embryonic Irf6-deficient studies epidermal wound-healing limiting in and 2006), al., orofacial et from (Ingraham anomalies perinatally healing wound die embryonic mice proper Irf6-deficient reflected for required likely is that Irf6 we cells. signal inflammatory 1B), Irf6 or (Fig. strong necrotic area following from a wound staining days of open background 11 presence the at the In before level shown). noted not expression epidermis, (data normal neoformed 1C). injury the (Fig. the epithelialization to 1B) in completed (Fig. returning We decreased injury just after mice. expression had day that 1 Irf6 adult at cells edge in in wound the and Irf6 adult at unwounded 1A), of of (Fig. keratinocytes in mice expression Irf6 al., of the presence et the (Le observed evaluate model to wound-healing murine 2012) excisional neoformed our the used in We and wounding edge excisional wound following the epidermis at present is Irf6 of migration RESULTS proper RhoA. by the we mediated for is assays, process required Irf6-dependent adhesion This is keratinocytes. and Irf6 of scratch-wound that culture demonstrated with using combination By embryos, Irf6-deficient wounds. in and wild-type embryonic from keratinocytes of primary healing the during nti td,w salse oefrIf nepithelialization in Irf6 for role a established we study, this In b 3 aadmntaeta r6i eurdfrpoe embryonic proper for required is wild-type Irf6 healing. These that of wound 1H,I). still (Fig. demonstrate wounds were Irf6 for wounds data deficient the the embryos whereas in h, open 1G,I), 24 significantly (Fig. were closed After removal were embryos limb the 1D–F). following (Fig. and immediately 1D,E), identical wounds (Fig. Irf6- their of indistinguishable and size wild-type were time-point, e10.5 embryos in developmental deficient forelimb this the removed At we and animals. embryos, protocol Irf6-deficient classic the late-stage modified the in phenotype hindlimb on rawscoe t2 o r6dfcetadwild-type and Irf6-deficient the for of h 95% 2E–G). 24 (Fig. versus at significantly respectively) (59% h closed keratinocytes, wound 24 was were scratch area and the wound keratinocytes Irf6-deficient 8 closing initial in 6, Irf6-deficient and similar delayed 0, Despite sizes, shown). at wild-type not wound taken data 2A–F; of were (Fig. Irf6 post-scratch images that monolayers Static hypothesis keratinocytes. confluent the wound scratch test a generated in To we migration, epidermal wound. to contributes the keratinocyte of across consists mainly migration wounds embryonic of closure The an closing wound in scratch delayed are keratinocytes Irf6-deficient fwl-yekrtncts(i.2)oine n moved and oriented 2H) (Fig. majority keratinocytes the that tracks wild-type centroid the 10-min from at of seen 2H,I be Fig. analysis can in image It presented intervals. are dynamic data two-dimensional The plots (2D-DIAS). a be perimeter system cells stacked using could and tracks by behavior individual centroid generated This the in as 2). time over 1, noted moved Movies material substrate, Irf6-deficient keratinocytes the the (supplementary to that wild-type or another revealed one period whereas to h adhere 18 to seemed an keratinocytes over min 5 the to every of microscopy imaging video live time-lapse perform used we keratinocytes, deficient nodrt ute netgt h irtr eeto Irf6- of defect migratory the investigate further to order In ora fCl cec 21)17 8024 doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal AC,1m DE,100 (D,E), mm 50 1 bars: (A–C), Scale (H,I). open embryos remained Irf6-deficient of wounds (G,I), the closed whereas largely h, were 24 wounds After wild-type (F). animals Irf6-deficient wild- and between type the identical of was size wounds The original arrow). (white create wound to a sectioned were hl, (F,I). Forelimbs area hindlimb. wound of quantification embryos, e10.5 and (E,H) Irf6-deficient and wild-type (D,G) of images microscopic (D electron post-injury. (C) and days edge) white 7 (B, wound day the 1 indicates at arrowhead skin wounds back excisional adult and of (A) blue) (DAPI, and DNA (red) nuclear Irf6 for staining Immunofluorescent healing. for wound required embryonic is adult and during healing expressed wound is Irf6 1. Fig. h oiotlbakbr hwtemean; the show * bars black horizontal the P , nvitro in 0.05. cac sa.Iae taken Images assay. scratch m 2 GH.I n I, and F In (G,H). m )Scanning I) m nvitro in m 2841

Journal of Cell Science n h ifrnewshgl infcn ( significant highly was difference the and ieto change. direction 6 EERHARTICLE RESEARCH 2842 instantaneous The 2L). 0.39 (Fig. was keratinocytes cells Irf6-deficient of control velocity of angles that 2O, with (Fig. compared a+b+c+d, change direction (distance average and path keratinocytes. the wild-type total of addition, ( those the and In with decreased compared and significantly Irf6 2O for Fig. 2J) both deficient (distance Fig. were path in B, net 2K) from Fig. The illustrated to Methods. computed A as and were from Materials time, the change To in over direction described tracks. length, positions and path less-persistent net length centroid defects, path and these analyze total wound statistically net and contrast, the decreased quantify (small crawling, towards by persistent directions reduced progress random in 2I), in (Fig. resulting arrows), lamellipodia keratinocytes extended frequently Irf6-deficient the net towards made lamellipodia of cells wound. extension these preferential wound that the scratch by revealed the progress plots of Perimeter direction arrow). the (large in recordin arrows) video (small of persistently Analysis (J–N) gray. scratc in the shown of is direction plot the perimeter of indicates each plots panel in perimeter each perimeter stacked * of cell mean; and bottom final the tracks the show The centroid bars at 100 travel. 2D-DIAS-generated black bar: arrow of Horizontal Scale (H,I) large s direction (F). scratch-wounds. mean+s.e.m. The not then cellular the had keratinocytes. (A,B), indicate cells show (I) confluency arrows Irf6-deficient Data Irf6-deficient whereas to small time. (E), and grown scratch over (H) were the closure wild-type closed Cells wound had representative keratinocytes. keratinocytes keratinocytes. of (B,D,F) wild-type Irf6-deficient percentage h, Irf6-deficient of the 24 and directionality of By and (A,C,E) (C,D). speed wild-type tip reduced yellow of of a monolayers with because confluent wound scratch in a wounds of scratch closure Impaired 2. Fig. ...,apoiaeyhl hto idtp el (0.73 cells wild-type of that half approximately s.e.m.), c a infcnl nrae nteasneo Irf6 of absence the in increased significantly was ) P , P m , P .0,Fg 2M). Fig. 0.001, /i ( m/min , .0)i cells in 0.001) .5 *** 0.05; 6 6 P 0.054), 0.023; , a .0 (Student’s 0.001 , b n eue pe nclslcigIf,sgetn htIf is Irf6 that wounds scratch suggesting keratinocyte vitro Irf6, of in healing lacking efficient cells the for in necessary speed migration directional reduced deficient 2N, and a (Fig. indicate cells results wild-type our of Collectively, that was than lower keratinocytes times Irf6-deficient three of persistence the Consequently, eaioye Fg B,sgetn htiiilclua adhesion the cellular Irf6-deficient in initial and that differences wild-type suggesting detect 3B), between not (Fig. cells did keratinocytes adherent we on of plastic), cells number (fibronectin, and adherent substrates of IV other on number collagen that the with extracellular in compared of increase laminin-332 type an investigated the and Despite on further dependent wild-type matrix. was We in between outcome in this difference 3A). keratinocytes whether (Fig. no Irf6 showed adhered keratinocytes of data of Irf6-deficient Our role plating. number the the after to the h of adhesion 1 understanding cellular at investigated substrate our we further migration, keratinocyte to order In the of matrix independent extracellular is size cellular Irf6-dependent t ts) O ehdfrcluaigntpt egh oa ahlnt and length path total length, path net calculating for Method (O) -test). . ora fCl cec 21)17 8024 doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal AF tl eodn of recording Still (A–F) m .()Quantification (G) m. on,and wound, h gof P cratched , 0.001). nvitro in nvitro in

Journal of Cell Science EERHARTICLE RESEARCH ysann ihpalii,amre fplmrzdactin polymerized of marker a phalloidin, with staining by hypothesize to cytoskeleton. us actin leading the 4B), regulates Irf6 (Fig. that roundness by cell accompanied was in it increase that found an we We and 4A), 2012). during (Fig. cells closure Irf6-deficient al., scratch in et size cellular occurrence (Biggs in increase the transition this to confirmed mesenchymal due to not is epithelial this of and cultures, for wild-type grown larger than contain when cells h keratinocytes Irf6-deficient that, 1 of at reported cultures size days, previously cellular several have in increase We an plating. showed after 3 Fig. in presented of Data amount the and RhoA cytoskeleton active actin the Irf6. regulates Irf6 of of potentially absence spreading by the cytoskeleton. the fashion actin restrict the in to substrate-independent appeared regulating acts a plating staining, Irf6 in after that phalloidin keratinocytes suggest h data 1 by our Thus, at identified prominent as more However, fibers, 3C,D). between the (Fig. differences stress keratinocytes and show actin Irf6-deficient not cell and did the component complex, wild-type the adhesion a between focal connect vinculin, the Irf6-deficient contact for of which Immunostaining of matrix. point to spreading, extracellular the the to cellular intrinsic cytoskeleton of and independent is adhesion is size but the supporting cell shown), matrix, keratinocytes. larger not the (data extracellular IV that collagen with hypothesis compared on the cells change plated Irf6-deficient adherent not cells of did size the keratinocytes or wild-type number by extracellular The the produced preformed changed. but on matrix 3E). not keratinocytes substrate, (Fig. was Irf6-deficient other mutant of laminin- any plating and counterparts on on wild-type plated between plated wild-type cells difference when with on spread their compared more grown 332 significantly were cells than cells were of Interestingly, Irf6 spread exception for We the more deficient of With 3D). keratinocytes collagen area fibronectin, laminin-332. fibronectin, versus plastic, cellular on and the plating 3C of (Fig. IV measuring h 1 cells by after keratinocytes observation cells wild-type this Irf6-deficient than that quantified observed larger h 1 consistently at were However, we Irf6. plating, of independent after is matrix extracellular the to efrtivsiae h atr fatnsrs ie arrangement fiber stress actin of pattern the investigated first We cell–matrix in involved structures cellular are adhesions Focal xeiet.Tecmie aasoe htclue fIrf6- of cultures that independent showed six data across combined 50.6% to The 24.1% experiments. stress from varied prominent prominent of with fibers cells those stress of more percentage with The 4C,D). observed compared (Fig. keratinocytes cells wild-type We Irf6-deficient the 1977). in fibers al., et (Wehland Fg I,tettlpt Fg J,teisatnosvelocity path instantaneous net the Irf6-deficient the of 4K) 4J), rescued (Fig. (Fig. persistence the inhibitor path consequently, and, total ROCK 4L) no (Fig. the the had 4I), Y27632 of (Fig. as However, that 4A,B). presence showed inhibitor, (Fig. shape the Irf6- results and ROCK size cellular Our and on the effect 2. significant wild-type Fig. with for from treated described data keratinocytes and microscopy deficient formation video fiber time-lapse stress and controlling active between thus migration. balance keratinocyte the RhoA, regulates Irf6 inactive that these together, Irf6- indicate Taken 4H). and data (Fig. wild-type groups two both the between h, difference significant statistically 18 no with After closed, 80% Y27632. were scratches deficient of presence the keratinocytes scratchedin of we partial monolayers consequences, confluent this Irf6-deficient functional and whether our had wild-type phenotypic test fibers stress To furthering the of RhoA. rescue regulates keratinocytes, rescued a Irf6 Irf6-deficient that partially yet hypothesis data fibers, ROCK of These stress 4F,G). blocking characteristics (Fig. of cultures that prominence both suggest in the observed in was reduction reduction greater Rho- exhibited a cultures a keratinocyte were Irf6-deficient keratinocytes Irf6-deficient Y27632. ROCK, with and treated wild-type blocked Both 1996). we al., (Leung RhoA et RhoA, of effector downstream on and kinase protein associated dependent Irf6-deficient were in observed healing cells scratch-wound in delays the and in Irf6 RhoA. for through role fibers a stress confirm regulating results compared negatively These keratinocytes cells. we wild-type Irf6-deficient of the in that in RhoA, RhoA, increase with RhoA an observed active of GTP-bound We of 4E). for activity amount (Fig. RhoA assay of the form precipitation active regulates the affinity Irf6 an test to that performed order In hypothesis 2001). Burridge, the inhibition and an (Arthur migration to cellular leading RhoA-GTPase, of of an activity with the associated in previously elevation been has assembly fibers premature stress cytoplasmic A prominent cells. of wild-type with of cells cultures than more fibers times stress 1.77 had keratinocytes deficient nodrt ute netgt h feto 262 eanalyzed we Y27632, of effect the investigate further to order In fibers stress of prominence increased the whether determine To ora fCl cec 21)17 8024 doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal aai n hwtema+... *** mean+s.e.m.; the show mean; E the and NS show B A in in data bars on black keratinocytes Horizontal (Irf6-deficient laminin-332). 1211 to keratinocytes fibronectin) (Irf6-deficient on 40 of from number varied The analyzed cells matrices. after extracellular h different 1 on at plating measured was keratinocytes Irf6-deficient DAPI 20 with bars: labeled Scale is (blue). DNA Irf6-deficient Nuclear and keratinocytes. (C) (D) phalloidin wild-type and of (green) staining vinculin (red) of determined. images was Confocal field (B) (C,D) per matrices cells per extracellular of cells different number of on average number or The (A) later. field h microscopic 1 laminin-332 fixed and and (CollIV) (Lam) IV (Pl), collagen plastic (FN), on fibronectin plated were keratinocytes of deficient substrate. independent matrix is extracellular size the cellular Irf6-dependent 3. Fig. P . .5(Student’s 0.05 t -test). m .()Teae fwl-yeand wild-type of area The (E) m. idtp n Irf6- and Wild-type P , 0.001; 2843

Journal of Cell Science EERHARTICLE RESEARCH ttepoenlvli uaeu ise rme75Irf6-deficient 2844 e17.5 from We tissues cutaneous Irf6. expression in of Arhgap29 level in protein absence decrease the the the at and non- in presence a showed the expression it at confirmed and reduced expressed GAPs other but was the than it significant skin GAP because the Irf6-deficient in candidate levels We higher a the decreased 2006). as al., in et Arhgap29 (Ingraham either ones identified expression wild-type the GEF to for relative samples increased to skin, or wild-type compares expression embryonic which data, levels, and RhoA microarray Irf6-deficient regulates our GAPs Irf6 searched (Cherfils how – identify we respectively To activity RhoA, 2013). RhoA Zeghouf, activating and regulate and inactivating proteins GEFs, of activity classes RhoA modulate Two to Arhgap29 and regulates size Irf6 cellular rescue reduced not does ROCK and but Irf6, migration that shape. lacking cells directional indicate in deficient speed results rescues our inhibitor Collectively, keratinocytes. rgp9 e euao fRo ciiy isdwsra of that downstream evidence lies activity, provide keratinocytes. RhoA the data in of Irf6 in These regulator key reduced 5D,E). a (Fig. were Arhgap29, of Irf6 expression of alteration Arhgap29 absence apparent cells, keratinocytes. of protein Irf6-deficient the levels no The and of However, with wild-type cytoplasm shown). pattern, between the localization punctate not within keratinocytes a perinuclear (data embryonic displaying be fibroblasts murine to appeared and in culture, in 5D,E) detected cells 5C). (Fig. In (Fig. was compartment. dermal with analysis Arhgap29 the epidermis, in the blot expression throughout some expressed immunostaining western mainly by was Arhgap29 and tissues wild-type 5A,B) (Fig. with compared embryos nrhme l,20) edmntaeta r6at sa as acts Irf6 2012; that al., the demonstrate inhibits Irf6 et that we show We (Biggs migration. 2006), keratinocyte model of al., regulator murine et Irf6-deficient Ingraham our Using DISCUSSION ora fCl cec 21)17 8024 doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal aea hs rsne nFg 2. the Fig. are in Y27632 presented without those samples as for same data the that Note ** cac ons aaso h ensem;* mean+s.e.m.; of the healing show the Data of wounds. recordings scratch video of Analysis (I–L) eaioye rw iho ihu 262( Y27632 without or Irf6-deficient with and grown wild-type keratinocytes scratch of of cultures Percentage in (H) closure RhoA. for Irf6-deficient probed and keratinocytes wild-type on RhoA GTP-bound 20 stress bar: arrows, Scale white (blue). fibers. actin; DAPI cortical with arrowheads, labeled White is DNA with Nuclear or Y27632. (C,D) (F,G) without Irf6-deficient grown and keratinocytes (C,F) (D,G) wild-type and of (green) staining vinculin (red) the phalloidin of show images bars Confocal black (C,D,F,G) without Horizontal mean. IV. or collagen with on grown (Y) cells Y27632 on wound- performed Irf6- a assay and during healing wild-type analyzed of were (B) keratinocytes roundness deficient RhoA. and active (A) of area levels The increased actin to the lead in cytoskeleton changes Irf6-dependent 4. Fig. P , .1 *** 0.01; P , .0;**** 0.001; m .()Afnt rcptto sa for assay precipitation Affinity (E) m. P , .01(n-a ANOVA). (one-way 0.0001 n 5 P 3–5). nvivo In , 0.05; nvitro in ,

Journal of Cell Science EERHARTICLE RESEARCH omnfnto nrgltn eaioyemgainduring migration a keratinocyte share regulating both in although Grhl3-deficient Thus, function study), 2010). Rho common al., (this and et formation activity fiber (Caddy stress activity Rho in decrease a and display keratinocytes fibers prominence stress the whereas in of increase However, activity. an showed Rho display this keratinocytes and genes Irf6-deficient fibers of these stress support of of levels either In altered lack common migration. that a keratinocytes epidermal in hypothesis, function regulating IRF6 in al., and et pathway Garza GRHL3 la keratinocytes that (de human periderm suggesting zebrafish in the 2013), in IRF6 and for 2011) Finally, al., target 2008). et direct (Botti al., a et as Hislop identified 2010; was al., addition, et an (Caddy In closing wound in 2014). delayed were al., an keratinocytes close deficient et to fail Peyrard-Janvid Grhl3 humans, 2002; lacking embryos In al., 2006). et al., (Kondo et (Yu in mutations differentiation epidermal regulate and to like required proliferation because, is that grainy-head relevant factor transcription lack particularly a that encodes are Mice (Grhl3) models. 3 murine like few of a of reminiscent reminiscent migration. 1999), cell epithelial The al., in evolutionarily Irf6 stage. potential for et gastrula role a conserved suggesting (Zalik late cells, epithelial at at junctions mammalian cadherins embryo and cell–cell cytoskeleton the actin the of an contains rupture fish layer the enveloping in the irf6 a of to absence as The cell. moves led yolk layer the cover enveloping to a epithelial layer 2009), coherent al., the the et which (Sabel in epiboly during proper of dominant-negativeprocess undergo postulated to a failed defect previously irf6 with of form injected was migratory embryos Irf6 potential Zebrafish zebrafish. lacking an A cells and assay. epithelial assay scratch wound-healing keratinocyte culture embryo murine 2010). molecular al., et potential post-surgical (Jones in without of mutations a those likelihood with patients increased provides in observed complications This the for area migration. cellular rationale of increased slower level fibers, stress the and actin in regulating result of changes by formation molecular increased RhoA These inactivator. GTPase RhoA a small Arhgap29, the of 50 activity bars: Scale N- (blue). in DAPI IV with collagen labeled on is grown DNA keratinocytes Nuclear (E) medium. wild- Irf6-deficient in e17.5 and (red) from Arhgap29 (D) for extracts type staining RIPA Immunofluorescent on (D,E) Arhgap29 skin. for embryonic sections. analyses cutaneous of blot (B) expression Western Irf6-deficient the (C) and in (A) decrease wild-type a embryonic to leads Irf6 of Arhgap29. absence The 5. Fig. u aaso easi on lsr sn ohan both using closure wound in delays show data Our h eeti eaioyemgaini h bec fIf is Irf6 of absence the in migration keratinocyte in defect The AB muoloecn tiigfrAha2 rd ne17.5 in (red) Arhgap29 for staining Immunofluorescent (A,B) IRF6 and GRHL3 aebt enietfe nVWS in identified been both have xvivo ex IRF6 on,adGrhl3- and wound, oprdwith compared nvitro in Irf6 nvitro in GRHL3 m , xvivo ex scratch m Grhl3 ua eaioye n nrae rlfrto Camne al., et of (Chapman immortalization proliferation the the increased to studies, and led separate inhibitor keratinocytes ROCK In human same 2001). the of Burridge, addition extensive and the (Arthur a t that previously to contribute of evidence cells mutant addition further in fibers The providing RhoA. thus migrato active keratinocytes, the in rescued inhibitor increase ROCK an shown), not by data group, promin accompanied wild-type the with with keratinocytes compared cells group Irf6-deficient the deficient more in find fibers to times surprised stress (1.77 not prominent more were of are we presence 1981), fibers inhibit (Burridge, stress and migration 1979) As Rees, cell and distance. 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Journal of Cell Science EERHARTICLE RESEARCH eepromda ecie rvosy(ig ta. 02 ee l,2012). al., 2846 et Le immunostaining 2012; al., and et sections (Biggs previously Serial described paraformaldehyde. as performed wounds 4% and were post-wounding, in days 11 and fixed 7 4, were 8–12-week-old 1, ( at of Animals euthanized 2012). back were al., group) et the per (Le on previously described performed as animals, were wild-type biopsies punch 6-mm Two vivo In Review Use and Care Animal distinct Two the Iowa. of to University according the at for Form cared were mice All Mice METHODS AND available. MATERIALS readily provide are and inhibitors processes Rho biological as both options, complications have therapeutic on studies new impact these wound-healing to Furthermore, potential VWS. the the with a patients offer for in studies observed these mechanism slowed development, palatal and processes molecular and formation the healing fiber to wound essential stress of is in migration keratinocyte increase As an migration. to Irf6 of of lead loss upstream the would Thus, acts RhoA. active Irf6 regulate negatively 6). to Arhgap29 (Fig. E-cadherin 2009). migration al., altered et keratinocyte (Richardson regulating periderm that mice oral Irf6-deficient the note of in epithelium defect to a oral to the interesting due in is observed is it expression cell– E-cadherin-mediated is adhesion, in Irf6 of signaling cell role the investigated RhoA Although not catenin 2000). have p120 al., we et Particularly, by (Anastasiadis inhibition contacts intercellular cell–cell its 1997). nascent of and affects clustering, al., site cadherin the for et necessary at the (Braga and establishment E-cadherin junction their the for activity of Rac stabilization and Rho proper migration. require cellular which in Irf6- adhesions, delay that disassembling a possibility to in are lead the defective would out cells are the rule from the cells adhesions cannot sever deficient which to we ability with Therefore, their or matrix. strength matrix the our the to of attached None for either. h tested 48 experiments after adhesion cell–matrix in differences migration. negatively promote also to could E-cadherin IRF6 regulate kinase migration. protein IRF6-dependent and Rho-associated rescues fibers the ROCK to stress Blocking leading of migration. decreased, prominence cellular are inactive increased impaired levels an RhoA, Arhgap29 to of IRF6, RhoA activity of returning increased absence thus GDP, the to In GTP state. of hydrolysis the promotes keratinocyte regulating pathway migration. Irf6-dependent An 6. Fig. ecie rvosy(ig ta. 02 nrhme l,20) The 2006). (e)0.5. al., day embryonic et as designated Ingraham was 2012; plug copulatory al., a of et presence (Biggs previously described eoyigfrthe for Genotyping necagal ooti r6dfcetebys– embryos Irf6-deficient obtain to interchangeably -ahrni ao opnn fahrn ucin,which junctions, adherens of component major a is E-cadherin nsmay ee ehv dniidanvlrl o r6in Irf6 for role novel a identified have we here, summary, In xiinlwudhealing wound excisional R6rgltsAha2,aGPs-ciaigpoenthat protein GTPase-activating a Arhgap29, regulates IRF6 Irf6 gt1 leeadthe and allele Irf6 del1/+ Irf6 uatsriswr used were strains mutant leewspromdas performed was allele Irf6 gt1/+ and Irf6 n 5 del1/+ 4–6 . rmtedri n nuae n02%tysn(ic Invitrogen, (Gibco 37 trypsin at 0.25% min in 20 for incubated CA) and Carlsbad, dermis the from idtp n r6dfcetkrtncts n ttciae were images static and keratinocytes, both Irf6-deficient of monolayers confluent and in tip P200 wild-type a with generated were Scratches vitro In (Roche II Dispase U/ml 4 5 at with IN) incubated Indianapolis, was Diagnostics, embryos e17.5 from Skin culture Keratinocyte 37 at O oven 95% Hybridization with Micro h Autoblot 12 Bellco 1% every a and with in at experiment placed gassed filter- supplemented were and the of vials NaCl of ml The beginning serum]. 4 (w/v) rat the containing 0.9% fresh tube part The parts 1 conical blades. penicillin-streptomycin, [3 a mm 4 in medium and with placed sterilized scissors sacs the then pregnant using wound, amniotic with were amputated embryonic their embryos from was the 1979), of generate bud To forelimb out removed placentae. Cockroft, left dissected their were to were and connected They previously left Embryos e10.5. New described at follows. 1995; females as as Martin, performed modifications was and healing (McCluskey wound Embryonic vivo Ex eaioye psae2 150cells/cm 21,500 2, (passage Keratinocytes assay adhesion Cell-substrate length, period. centroid path 18-h the from an line n over net a frame intervals drawing in by 10-min computed length, at was velocity position computed Instantaneous centroid path were cell change the Total direction from and persistence beta- images. velocity, to converted instantaneous images were replacement and phase-contrast feature from trace spline outlines manual the cell Wessels using accurate 1998; obtained 2D-DIAS Voss, were Briefly, and using 2009). 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Journal of Cell Science h saswr efre sdsrbdpeiul Rne l,1999). al., et (Ren previously described as performed were assays Rho pulldown RhoA Active cells/cm were 21,500 cells averaged. of and Two Similarly, density counted duplicate. 2012). a were in at al., cells performed the plated et were and (Biggs experiments taken, were Three previously well described per images as buffer, Giordano ARTICLE RESEARCH aoaoisfo h nvriyo oawopoie rs a eu.Abgthank big A serum. rat as fresh well provided as who IA), Iowa City, of Iowa University Iowa, the Microscopy of from (University Central laboratories Rhea the Lindsey Keck at and W.M. Walters Facility the Katherine Research at Facility, Wessels authors Analysis Deborah The Image of support. Dynamic assistance unconditional technical his for the Murray acknowledge Jeff acknowledge authors The Statistical Acknowledgements replicates. biological indicated three as legends. study, least figure each the for at tests in appropriate of with performed means was analysis the are Data Statistics NY). microscope (Thornwood, 710 software LSM 2009 Zeiss ZEN a and E800 using NY) confocal (Thornwood, acquired For Eclipse merged. were and Nikon images pseudocolorized using microscopy, were MI). a camera images Heights, white CCD Sterling with and slider Instruments, Black RT (Diagnostic viewed SPOT software a Advanced were with Spot acquired Images and NY) stain. with (Melville, incubated as nuclear and used PBS was a (DAPI) in were 4,6-diamidino-2-phenylindole washed cells antibodies. blocking CA), antibodies, secondary After Burlingame, primary 1996). Laboratories, with al., (Vector incubated et serum (Michel goat 3% previously with coverslips described collagen-IV-coated as on fixed grown and were 1 passage (GE at Keratinocytes ECL system Microscopy detection chemiluminescent the Healthcare). was horseradish- detection with with antigen performed antibodies, incubation and IgG After milk secondary antibodies. dried peroxidase-conjugated nonfat primary 10% conditions. with in (Bio blocked incubated membranes denaturing CA), fluoride Hercules, 10% under Laboratories, polyvinylidene Rad on onto gels transferred separated were were SDS-PAGE Proteins protein (Invitrogen) of for amounts Bis-Tris used Equal was preparation. buffer protein extraction (RIPA) assay Radioimmunoprecipitation analysis and Protein NJ) (Piscataway, Healthcare respectively. GE Biotechnology, Cruz from (clone Santa were CA). RhoA Cruz, antibodies Biologicals horseradish-peroxidase-conjugated (Santa secondary against Biotechnology anti-rabbit-IgG Novus Cruz and antibody Santa from Anti-mouse-IgG from monoclonal obtained Rhodamine- obtained polyclonal was Mouse 26C4) Rabbit was and CO). Sigma. Arhgap29 vinculin (Littleton, from against obtained against antibody were antibodies phalloidin conjugated monoclonal Mouse Antibodies 0.5% SDS, 0.1% NaCl, MgCl mM mM 10 500 X-100, 7.6, Triton lysis on pH 1% in immobilized deoxycholate, Tris-HCl sodium lysed was mM (50 were rhotekin buffer Cells of beads. domain glutathione-S-transferase-conjugated RhoA-binding the Briefly, aho MF eppi n poii) n qa mut fcl lysate 4 cell of 30 at with amounts beads equal with and incubated aprotinin), were and leupeptin PMSF, of each bandfo im S oi,M)adue tacnetainof concentration a at used and MO) Louis, (St was 10 Sigma Y27632 Three inhibitor from antibody. ROCK RhoA-specific obtained performed. with were (Invitrogen). probed gels experiments Bis-Tris then independent 10% were on membranes run and The blotting western for prepared oesiswr hnsandwt iclnadphalloidin. The and later. vinculin min with 60 stained v/v) then (75:25, were methanol:acetone coverslips in fixed and coverslips ie n7%ethanol. 70% in fixed m ˚ .Clswr nuae nY73 rDS oto o 4hand h 24 for control DMSO or Y27632 in incubated were Cells M. ihrtto.Ttllst n edcnuae yaewere lysate bead-conjugated and lysate Total rotation. with C m fGTbudroei o 0min 30 for rhotekin GST-bound of g 2 noclae-Vcae glass collagen-IV-coated onto 2 n 10 and m g/ml od,K n aiuh,T. L., Taniguchi, and S. K. King, Honda, S., Vasudevan, A., Auden, B., S. Ting, J., Caddy, R., N. Hislop, ae,B,Bcebc,J .adFeka,P. Fleckman, and R. J. Bickenbach, B., Hager, esa,S .adRde,A J. A. Ridley, and J. S. Heasman, rhr .T n urde K. Burridge, and Crawford, T. J., W. D. Mariner, Arthur, A., M. Thoreson, Y., S. Moon, Z., P. Anastasiadis, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139246/-/DC1 online available material Supplementary material Supplementary months. 12 after release for the PMC by in initiated Deposited Resource Health. National of a Institutes Iowa, National of University the at Studies Developmental Bank Hybridoma the by supported [grant was professions health D.R.S. 5T35HL007485-34]; the number in students for training short-term National the for Health from grant of Institutes a by supported was Foundation R.L.N. Science K.A.D.]. National to 1120478 the number Health [grant and B.C.S.]; of Institutes and National M.D. the to AR035313 from number funding [grant by supported partially was work This Funding wrote M.D. and B.C.S. K.A.D., L.C.B., data; D.R.S., paper. the K.A.D., the analyzed R.L.N., M.D. L.C.B., and experiments; S.K. performed D.F.L., M.D. and R.L.N. L.C.B., contributions Author interests. competing no declare authors The interests Competing ulu,C,Gri-aa .adBrig,K. Burridge, and R. Garcia-Mata, C., Guilluy, P., Ostano, E., Calautti, A., Vignano, Di Tommasi A., Hiou-Feige, M., Grossi, M. Zeghouf, and J. Cherfils, A. N. Hotchin, and A. Hall, M., L. Machesky, M., V. Braga, J. C. Arpey, and L. C. Baum, utr ehiu n nrwLda Uiest fIw)frteueo his of use the for Iowa) wound of embryonic (University the Lidral us microscope. teaching Andrew for and UK) technique Bristol, of culture (University Martin Paul to you el az,G,Shefat,J . unad . akd . erte,J L., J. Weirather, A., Mankad, M., Dunnwald, R., J. Schleiffarth, G., Garza, la de A. P. Coulombe, A. D. Rees, and R. J. Couchman, A. A. McBride, and R. Schlegel, C., Meyers, X., Liu, S., Chapman, Meo, De S., Galanti, V., Pinetti, B., Marinari, F., M. Moretti, Dunnwald, G., Spallone, and E., Botti, C. B. Schutte, L., Rhea, C., L. Biggs, ad,J,Wlnwk,T,Drd,C,Dokn . ig .B,Za,Q,Rank, Q., Zhao, B., S. Ting, S., Dworkin, C., Darido, T., Wilanowski, J., Caddy, K. Burridge, 644-658. iercposadctslcptenrcgiinreceptors. pattern-recognition cytosolic and receptors like migration. epidermal in M. roles S. Jane, coordinate and play 263-272. M. J. Lmo4 Cunningham, and E., Grhl3 J. Visvader, J., G. Lindeman, noterfntosfo nvv studies. vivo in from functions their into keratinocytes. epidermal murine .C,Zeg .adRyod,A B. A. Reynolds, and Y. catenin. Zheng, C., H. nte oilnetwork? KyoT1/2 social of another up-regulation with coupled signaling expression. (FHL1) P. Rho/CRIK G. by Dotto, and differentiation G. Chiorino, S., Lee, and protrusion membrane promoting by migration polarity. and spreading cell regulates od,G,Bthr . asu,T . os,Y . uaaa .F tal. by et periderm 3. F. the Grainyhead-like C. of of Fukazawa, differentiation A., expression promotes Y. 6 activating Kousa, factor A., regulatory T. Interferon Mansour, (2013). S., Butcher, G., Bonde, inhibitor. kinase GDIs. and Rho GAPs, a by immortalized Invest. efficiently Clin. J. are keratinocytes Human 138-147. cadherin- of establishment the for required are Rac contacts. cell-cell and dependent carcinomas. Rho cell GTPases squamous in USA activity Sci. suppressor Acad. Natl. tumor Castrignano Proc. F., exhibits Ganci, IRF6 F., factor Nicola, De D., P. differentiation. keratinocyte for Dermatol. sufficient, Invest. not J. but necessary, is 6 factor regulatory events. molecular 686. and discussion cellular with correlation rmcikhatepat:cagsi dein oooinadgot,adin discovery. and growth, fibronectin. and and locomotion actomyosin adhesion, of in distribution changes the explants: heart chick from pathway. signaling polarity cell planar the by al. regulated et A. is T. Papenfuss, repair S., Srivastava, A., Auden, G., ora fCl cec 21)17 8024 doi:10.1242/jcs.139246 2840–2848 127, (2014) Science Cell of Journal o.Bo.Cell Biol. Mol. a.Cl Biol. 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Journal of Cell Science ngt .S,Shte .C,Jag .adDxn .J. M. Dixon, and R. Jiang, C., B. Schutte, S., A. Knight, err-avd . ele .J,Kua .A,Sih .L,Dnwl,M., Dunnwald, L., T. Smith, A., Y. Kousa, J., E. Leslie, M., Peyrard-Janvid, oe,J . aay .W,Boks .T,Why .L,LHuex J., L’Heureux, L., G. Wehby, T., J. Brookes, W., J. Canady, L., J. Jones, Z., Wang, R., Karlsson, A., Basse, E., Pedersen, K., Peyrollier, J., B., K. Jackson, Trout, S., Sajan, B., Yang, S., Kondo, A., Kinoshita, R., C. Ingraham, ARTICLE RESEARCH 2848 M., F. Watt, S., Broad, J., D. Radford, H., V. Robertson, S., Lax, R., McMullan, P. Martin, and J. McCluskey, L. Lim, and E. Manser, Q., X. Chen, T., Leung, M., Cooper, S., Bullard, K., Schuette, C., L. C., Biggs, A., M. B. Mansilla, J., Schutte, E. Leslie, L., Rhea, F. M. C., Carlier, and L. C. Clainche, Le Biggs, J., Morrison, S., R., A. Naridze, Knight, C., M., B. Le, Bjork, J., R. Richardson, C., B. Schutte, S., Kondo, B. K. Yancey, and E. R. Burgeson, P., M. 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