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RESEARCH ARTICLE

Interferon regulatory factor 6 regulates keratinocyte migration

Leah C. Biggs1,2, Rachelle L. Naridze1, Kris A. DeMali3, Daniel F. Lusche4, Spencer Kuhl4, David R. Soll4,Brian C. Schutte5 and Martine Dunnwald1,2,*

ABSTRACT

Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation

and differentiation. In this study, we tested the hypothesis that

Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos

at 10.5 days post-conception failed to close their wound compared

with wild-type embryos. In vitro, Irf6-deficient murine embryonic

keratinocytes were delayed in closing a scratch wound. Live imaging of

the scratch showed deficient directional migration and reduced speed

in cells lacking Irf6. To understand the underlying molecular

mechanisms, cell–cell and cell–matrix adhesions were investigated.

We show that wild-type and Irf6-deficient keratinocytes adhere similarly

to all matrices after 60 min. However, Irf6-deficient keratinocytes were

consistently larger and more spread, a phenotype that persisted during

the scratch-healing process. Interestingly, Irf6-deficient keratinocytes

exhibited an increased network of stress fibers and active RhoA

compared with that observed in wild-type keratinocytes. Blocking

ROCK, a downstream effector of RhoA, rescued the delay in closing

scratch wounds. The expression of Arhgap29, a Rho GTPase-

activating protein, was reduced in Irf6-deficient keratinocytes. Taken

together, these data suggest that Irf6 functions through the RhoA

pathway to regulate cellular migration.

KEY WORDS: Interferon regulatory factor 6, Migration,

Keratinocytes

INTRODUCTIONCutaneous wound healing requires the coordination of

inflammation, epithelialization, angiogenesis and dermal repair

(Baum and Arpey, 2005). Epithelialization is ultimately dependent

on the migration, proliferation and differentiation of keratinocytes

(Coulombe, 2003). The growth and differentiation of keratinocytes

is tightly regulated by transcription factors, with the transcription

factor interferon regulatory factor 6 (Irf6) playing a crucial role

(Biggs et al., 2012; Botti et al., 2011; Ingraham et al., 2006).

Irf6 belongs to the IRF family of transcription factors, which

mediate the interferon response after viral infection (Honda and

Taniguchi, 2006). In contrast to most IRFs, Irf6 is essential during

embryogenesis. Mice lacking Irf6 exhibit perinatal lethality, as

well as limb, craniofacial and epidermal anomalies (Ingraham

et al., 2006; Richardson et al., 2006). In humans, mutations in

IRF6 cause Van der Woude syndrome (VWS) and popliteal

pterygium syndrome, two orofacial clefting disorders (Kondo

et al., 2002). Interestingly, patients with VWS were more likely to

have wound complications following corrective cleft surgery than

patients with non-syndromic cleft (Jones et al., 2010), consistent

with a role for IRF6 in wound healing. Although Irf6 is expressed

in suprabasal keratinocytes of the epidermis and plays a crucial

role in epidermal differentiation in vivo and in vitro (Biggs et al.,

2012; Ingraham et al., 2006), its function in keratinocyte

migration is currently unknown.

Cellular migration is a highly coordinated biological process

that includes the assembly of cell–cell and cell–matrix contacts

followed by the disassembly of older ones (for a review, see

Vicente-Manzanares et al., 2005). The coordination of migration

and the force required to achieve it is driven by the reorganization

of the actin cytoskeleton (for a review, see Le Clainche and

Carlier, 2008). The actin cytoskeleton normally provides dynamic

structure and organization to the cell but, in the event of

migration, this cellular scaffold reorganizes the cellular contents,

drives the formation of lamellipodia and filopodia (two cellular

protrusions defining the leading edge of a cell), and disassembles

to retract the tail of the cell (Le Clainche and Carlier, 2008;

Vicente-Manzanares et al., 2005). Migratory cues are diverse, and

sensors of such cues include clusters of integrins, located on

cellular protrusions, that assemble and disassemble to allow

migration (Zaidel-Bar and Geiger, 2010). Simultaneously, E-

cadherin-mediated cell–cell adhesions form initial contacts with

adjacent cells that subsequently evolve into linear adhesions

(Vasioukhin et al., 2000). The cytoskeleton requires integrins and

cadherins for information about the environment, and, in turn, the

contraction of actin is necessary for the assembly of the adhesions

that these proteins form with the substrate and with other cells

(Vasioukhin et al., 2000).

Members of the Rho family of small GTPases are the central

regulators of actin cytoskeleton dynamics. GTPases cycle

between GTP-bound (active) and GDP-bound (inactive) forms

through the control of guanine nucleotide exchange factors

(GEFs, activating) and GTPase activating proteins (GAPs,

inactivating) (Guilluy et al., 2011; Heasman and Ridley, 2008;

Van Aelst and D’Souza-Schorey, 1997). Of particular interest is

RhoA, the main small GTPase responsible for assembling stress

fibers that are anchored at adhesion complexes and that support

the cell contraction necessary for translocation (Ridley and Hall,

1992). In vitro studies demonstrate a role for RhoA GTPase in

keratinocyte differentiation (Grossi et al., 2005; McMullan et al.,

2003). In vivo, however, RhoA has been found to be dispensable

for epidermal differentiation but necessary for directed

keratinocyte migration (Jackson et al., 2011). RhoA activation

is also required for the formation of TGFb3-induced stress fibers

and for mediating TGFb3 signaling during palatogenesis

(Kaartinen et al., 2002). Additionally, we identified Arhgap29,

1Department of Pediatrics, The University of Iowa, Iowa City, IA 52242, USA.2Interdisciplinary Graduate Program in Genetics, The University of Iowa, IowaCity, IA 52242, USA. 3Department of Biochemistry, The University of Iowa, IowaCity, IA 52242, USA. 4Developmental Studies Hybridoma Bank, Department ofBiology, The University of Iowa, Iowa City, IA 52242, USA. 5Departments ofMicrobiology and Molecular Genetics and of Pediatrics and HumanDevelopment, Michigan State University, East Lansing, MI 48824, USA.

*Author for correspondence (martine-dunnwald@uiowa.edu)

Received 31 July 2013; Accepted 10 April 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 2840–2848 doi:10.1242/jcs.139246

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a GEF with high affinity for RhoA, as a novel cleft candidategene downstream of Irf6 (Leslie et al., 2012; Saras et al., 1997).

Because Irf6 is required for proper palatogenesis (Ingraham et al.,2006; Knight et al., 2006) and is a downstream effector of TGFb3(Knight et al., 2006; Xu et al., 2006), we hypothesize that Irf6regulates the actin cytoskeleton in keratinocytes and alters

cellular migration.In this study, we established a role for Irf6 in epithelialization

during the healing of embryonic wounds. By using culture of

primary keratinocytes from wild-type and Irf6-deficient embryos,in combination with scratch-wound and adhesion assays, wedemonstrated that Irf6 is required for the proper migration of

keratinocytes. This Irf6-dependent process is mediated by RhoA.

RESULTSIrf6 is present at the wound edge and in the neoformedepidermis following excisional woundingWe used our excisional murine wound-healing model (Le et al.,2012) to evaluate the expression of Irf6 in adult mice. We

observed the presence of Irf6 in keratinocytes of unwounded adultmice (Fig. 1A), at the wound edge at 1 day after injury (Fig. 1B)and in cells that had just completed epithelialization (Fig. 1C).

Irf6 expression decreased in the neoformed epidermis, beforereturning to the normal expression level at 11 days followinginjury (data not shown). In the open wound area (Fig. 1B), we

noted the presence of a strong Irf6 signal that likely reflectedbackground staining from necrotic or inflammatory cells.

Irf6 is required for proper embryonic wound healingIrf6-deficient mice die perinatally from orofacial and epidermalanomalies (Ingraham et al., 2006), limiting wound-healing studiesin Irf6-deficient animals to the embryo. Well-established

embryonic wound-healing models have been described thatfollow epidermal closure after hindlimb resection at embryonicday (e)11.5 (McCluskey and Martin, 1995). Because of the severe

hindlimb phenotype in the late-stage Irf6-deficient embryos, wemodified the classic protocol and removed the forelimb in e10.5

animals. At this developmental time-point, wild-type and Irf6-deficient embryos were indistinguishable (Fig. 1D,E), and thesize of their wounds immediately following limb removal wereidentical (Fig. 1D–F). After 24 h, the wounds of wild-type

embryos were closed (Fig. 1G,I), whereas the wounds were stillsignificantly open in embryos deficient for Irf6 (Fig. 1H,I). Thesedata demonstrate that Irf6 is required for proper embryonic

wound healing.

Irf6-deficient keratinocytes are delayed in closing an in vitroscratch woundThe closure of embryonic wounds mainly consists of keratinocytemigration across the wound. To test the hypothesis that Irf6

contributes to epidermal migration, we generated a scratch woundin confluent monolayers of wild-type and Irf6-deficientkeratinocytes. Static images were taken at 0, 6, 8 and 24 hpost-scratch (Fig. 2A–F; data not shown). Despite similar initial

wound sizes, Irf6-deficient keratinocytes were significantlydelayed in closing the scratch wound (59% versus 95% of thewound area was closed at 24 h for Irf6-deficient and wild-type

keratinocytes, respectively) (Fig. 2E–G).In order to further investigate the migratory defect of Irf6-

deficient keratinocytes, we used time-lapse video microscopy to

perform live imaging of the in vitro scratch assay. Images takenevery 5 min over an 18 h period revealed that the Irf6-deficientkeratinocytes seemed to adhere to one another or to the substrate,

whereas wild-type keratinocytes moved as individual cells(supplementary material Movies 1, 2). This behavior could benoted over time in the centroid tracks and stacked perimeter plotsgenerated by using a two-dimensional dynamic image analysis

system (2D-DIAS). The data are presented in Fig. 2H,I at 10-minintervals. It can be seen from the centroid tracks that the majorityof wild-type keratinocytes (Fig. 2H) oriented and moved

Fig. 1. Irf6 is expressed during adultwound healing and is required forembryonic wound healing.Immunofluorescent staining for Irf6 (red) andnuclear DNA (DAPI, blue) of adult back skin(A) and excisional wounds at 1 day (B, whitearrowhead indicates the wound edge) and7 days (C) post-injury. (D2I) Scanningelectron microscopic images of wild-type(D,G) and Irf6-deficient (E,H) e10.5 embryos,and quantification of wound area (F,I). hl,hindlimb. Forelimbs were sectioned to createa wound (white arrow). The size of theoriginal wounds was identical between wild-type and Irf6-deficient animals (F). After 24 h,wild-type wounds were largely closed (G,I),whereas the wounds of Irf6-deficient embryosremained open (H,I). Scale bars: 50 mm(A–C), 1 mm (D,E), 100 mm (G,H). In F and I,the horizontal black bars show the mean;*P,0.05.

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persistently (small arrows) in the direction of the scratch wound(large arrow). Perimeter plots revealed that these cells made netprogress by the preferential extension of lamellipodia towards the

wound. Irf6-deficient keratinocytes (Fig. 2I), by contrast,frequently extended lamellipodia in random directions (smallarrows), resulting in reduced persistent crawling, decreased net

progress towards the wound and less-persistent tracks. Toquantify and statistically analyze these defects, net path length,total path length and direction change were computed fromcentroid positions over time, as illustrated in Fig. 2O and

described in the Materials and Methods. The net path (distancefrom A to B, Fig. 2J) and the total path (distance a+b+c+d,Fig. 2K) were both significantly decreased (P,0.001) in cells

deficient for Irf6 compared with those of wild-type keratinocytes.In addition, the average direction change (Fig. 2O, angles a, band c) was significantly increased in the absence of Irf6

compared with that of control cells (Fig. 2L). The instantaneousvelocity of Irf6-deficient keratinocytes was 0.39 mm/min (60.023;6s.e.m.), approximately half that of wild-type cells (0.7360.054),and the difference was highly significant (P,0.001, Fig. 2M).

Consequently, the persistence of Irf6-deficient keratinocytes wasthree times lower than that of wild-type cells (Fig. 2N, P,0.001).Collectively, our results indicate a deficient directional migration

and reduced speed in cells lacking Irf6, suggesting that Irf6 isnecessary for the efficient healing of keratinocyte scratch woundsin vitro.

Irf6-dependent cellular size is independent of theextracellular matrixIn order to further our understanding of the role of Irf6 in

keratinocyte migration, we investigated cellular adhesion to thesubstrate at 1 h after plating. Our data showed no difference inthe number of adhered keratinocytes between wild-type and

Irf6-deficient keratinocytes (Fig. 3A). We further investigatedwhether this outcome was dependent on the type of extracellularmatrix. Despite an increase in the number of adherent cells on

laminin-332 compared with that on other substrates (fibronectin,collagen IV and plastic), we did not detect differences in thenumber of adherent cells between wild-type and Irf6-deficientkeratinocytes (Fig. 3B), suggesting that initial cellular adhesion

Fig. 2. Impaired closure of a scratch wound because of reduced speed and directionality of Irf6-deficient keratinocytes. (A–F) Still recording of in vitro

scratch wounds in confluent monolayers of wild-type (A,C,E) and Irf6-deficient (B,D,F) keratinocytes. Cells were grown to confluency (A,B), then scratchedwith a yellow tip (C,D). By 24 h, wild-type keratinocytes had closed the scratch (E), whereas Irf6-deficient cells had not (F). Scale bar: 100 mm. (G) Quantificationof the percentage of wound closure over time. Data show the mean+s.e.m. (H,I) 2D-DIAS-generated centroid tracks and stacked perimeter plots ofrepresentative wild-type (H) and Irf6-deficient (I) keratinocytes. The large arrow at the bottom of each panel indicates the direction of the scratch wound, andsmall arrows indicate cellular direction of travel. The final cell perimeter in each perimeter plot is shown in gray. (J–N) Analysis of video recording of in vitro

scratch-wounds. Horizontal black bars show the mean; *P,0.05; ***P,0.001 (Student’s t-test). (O) Method for calculating net path length, total path length anddirection change.

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to the extracellular matrix is independent of Irf6. However, at 1 hafter plating, we consistently observed that Irf6-deficient cellswere larger than wild-type cells (Fig. 3C versus 3D). We

quantified this observation by measuring the cellular area ofkeratinocytes after 1 h of plating on plastic, fibronectin, collagenIV and laminin-332. With the exception of cells grown onfibronectin, keratinocytes deficient for Irf6 were significantly

more spread than their wild-type counterparts (Fig. 3E).Interestingly, cells were more spread when plated on laminin-332 compared with cells plated on any other substrate, but the

difference between wild-type and mutant was not changed. Theplating of Irf6-deficient keratinocytes on preformed extracellularmatrix produced by wild-type keratinocytes did not change the

number or the size of adherent Irf6-deficient cells compared withcells plated on collagen IV (data not shown), supporting thehypothesis that the larger cell size is independent of the

extracellular matrix, but is intrinsic to Irf6-deficientkeratinocytes.

Focal adhesions are cellular structures involved in cell–matrixadhesion and cellular spreading, which connect the actin

cytoskeleton to the point of contact between the cell and theextracellular matrix. Immunostaining for vinculin, a componentof the focal adhesion complex, did not show differences between

wild-type and Irf6-deficient keratinocytes (Fig. 3C,D). However,stress fibers, as identified by phalloidin staining, appearedmore prominent at 1 h after plating in the absence of Irf6.

Thus, our data suggest that Irf6 acts to restrict the spreading ofkeratinocytes in a substrate-independent fashion by potentiallyregulating the actin cytoskeleton.

Irf6 regulates the actin cytoskeleton and the amount ofactive RhoAData presented in Fig. 3 showed an increase in cellular size at 1 h

after plating. We have previously reported that, when grown forseveral days, cultures of Irf6-deficient keratinocytes contain largercells than wild-type cultures, and this is not due to the occurrence

of epithelial to mesenchymal transition (Biggs et al., 2012). Weconfirmed this increase in cellular size in Irf6-deficient cells duringscratch closure (Fig. 4A), and we found that it was accompanied by

an increase in cell roundness (Fig. 4B), leading us to hypothesizethat Irf6 regulates the actin cytoskeleton.

We first investigated the pattern of actin stress fiber arrangementby staining with phalloidin, a marker of polymerized actin

(Wehland et al., 1977). We observed more prominent stressfibers in the Irf6-deficient keratinocytes compared with those ofwild-type cells (Fig. 4C,D). The percentage of cells with prominent

stress fibers varied from 24.1% to 50.6% across six independentexperiments. The combined data showed that cultures of Irf6-deficient keratinocytes had 1.77 times more cells with prominentstress fibers than cultures of wild-type cells. A premature assembly

of cytoplasmic stress fibers has been previously associated with anelevation in the activity of RhoA-GTPase, leading to an inhibitionof cellular migration (Arthur and Burridge, 2001). In order to test

the hypothesis that Irf6 regulates the activity of RhoA, weperformed an affinity precipitation assay for GTP-bound RhoA,the active form of RhoA (Fig. 4E). We observed an increase in the

amount of active RhoA in Irf6-deficient keratinocytes comparedwith that of wild-type cells. These results confirm a role for Irf6 innegatively regulating stress fibers through RhoA.

To determine whether the increased prominence of stress fibersand the delays in scratch-wound healing observed in Irf6-deficientcells were dependent on RhoA, we blocked ROCK, a Rho-associated protein kinase and downstream effector of RhoA (Leung

et al., 1996). Both wild-type and Irf6-deficient keratinocytes weretreated with Y27632. Irf6-deficient keratinocyte cultures exhibiteda greater reduction in the prominence of stress fibers, yet a

reduction was observed in both cultures (Fig. 4F,G). These datasuggest that blocking ROCK partially rescued the phenotypiccharacteristics of Irf6-deficient keratinocytes, furthering our

hypothesis that Irf6 regulates RhoA. To test whether this partialrescue of stress fibers had functional consequences, we scratchedwild-type and Irf6-deficient confluent monolayers of keratinocytesin the presence of Y27632. After 18 h, both wild-type and Irf6-

deficient scratches were 80% closed, with no statistically significantdifference between the two groups (Fig. 4H). Taken together, thesedata indicate that Irf6 regulates the balance between active and

inactive RhoA, thus controlling stress fiber formation andkeratinocyte migration.

In order to further investigate the effect of Y27632, we analyzed

time-lapse video microscopy data from wild-type and Irf6-deficient keratinocytes treated with the ROCK inhibitor, asdescribed for Fig. 2. Our results showed that Y27632 had no

significant effect on cellular size and shape (Fig. 4A,B). However,the presence of the ROCK inhibitor rescued the net path(Fig. 4I), the total path (Fig. 4J), the instantaneous velocity(Fig. 4L) and, consequently, the persistence (Fig. 4K) of Irf6-deficient

Fig. 3. Irf6-dependent cellular size is independent ofthe extracellular matrix substrate. Wild-type and Irf6-deficient keratinocytes were plated on plastic (Pl),fibronectin (FN), collagen IV (CollIV) and laminin-332(Lam) and fixed 1 h later. The number of cells permicroscopic field (A) or average number of cells per fieldon different extracellular matrices (B) was determined.(C,D) Confocal images of vinculin (green) and phalloidin(red) staining of wild-type (C) and Irf6-deficient(D) keratinocytes. Nuclear DNA is labeled with DAPI(blue). Scale bars: 20 mm. (E) The area of wild-type andIrf6-deficient keratinocytes was measured at 1 h afterplating on different extracellular matrices. The number ofcells analyzed varied from 40 (Irf6-deficient keratinocyteson fibronectin) to 1211 (Irf6-deficient keratinocytes onlaminin-332). Horizontal black bars in A show the mean;data in B and E show the mean+s.e.m.; ***P,0.001;NSP.0.05 (Student’s t-test).

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keratinocytes. Collectively, our results indicate that ROCK

inhibitor rescues deficient directional migration and reducedspeed in cells lacking Irf6, but does not rescue cellular size andshape.

Irf6 regulates Arhgap29 to modulate RhoA activityTwo classes of proteins regulate RhoA activity – GAPs andGEFs, inactivating and activating RhoA, respectively (Cherfils

and Zeghouf, 2013). To identify how Irf6 regulates RhoA levels,we searched our microarray data, which compares wild-type toIrf6-deficient embryonic skin, for either decreased GAP

expression or increased GEF expression in the Irf6-deficientsamples relative to the wild-type ones (Ingraham et al., 2006). Weidentified Arhgap29 as a candidate because it was expressed at

higher levels in the skin than the other GAPs and it showed a non-significant but reduced expression in the absence of Irf6. Weconfirmed the presence and the decrease in Arhgap29 expressionat the protein level in cutaneous tissues from e17.5 Irf6-deficient

embryos compared with wild-type tissues by immunostaining

(Fig. 5A,B) and western blot analysis (Fig. 5C). In vivo,Arhgap29 was mainly expressed throughout the epidermis, withsome expression in the dermal compartment. In cells in culture,Arhgap29 was detected in murine embryonic keratinocytes

(Fig. 5D,E) and fibroblasts (data not shown). The proteinappeared to be perinuclear within the cytoplasm of the cells,displaying a punctate pattern, with no apparent alteration of

localization between wild-type and Irf6-deficient keratinocytes.However, levels of Arhgap29 expression were reduced in theabsence of Irf6 (Fig. 5D,E). These data provide evidence that

Arhgap29, a key regulator of RhoA activity, lies downstream ofIrf6 in keratinocytes.

DISCUSSIONUsing our Irf6-deficient murine model (Biggs et al., 2012;Ingraham et al., 2006), we demonstrate that Irf6 acts as aregulator of keratinocyte migration. We show that Irf6 inhibits the

Fig. 4. Irf6-dependent changes in the actincytoskeleton lead to increased levels of active RhoA.The area (A) and roundness (B) of wild-type and Irf6-deficient keratinocytes were analyzed during a wound-healing assay performed on cells grown with or withoutY27632 (Y) on collagen IV. Horizontal black bars show themean. (C,D,F,G) Confocal images of vinculin (green) andphalloidin (red) staining of wild-type (C,F) and Irf6-deficient(D,G) keratinocytes grown without (C,D) or with(F,G) Y27632. Nuclear DNA is labeled with DAPI (blue).White arrowheads, cortical actin; white arrows, stressfibers. Scale bar: 20 mm. (E) Affinity precipitation assay forGTP-bound RhoA on wild-type and Irf6-deficientkeratinocytes probed for RhoA. (H) Percentage of scratchclosure in cultures of wild-type and Irf6-deficientkeratinocytes grown with or without Y27632 (n53–5).(I–L) Analysis of video recordings of the healing of in vitro

scratch wounds. Data show the mean+s.e.m.; *P,0.05;**P,0.01; ***P,0.001; ****P,0.0001 (one-way ANOVA).Note that the data for samples without Y27632 are thesame as those presented in Fig. 2.

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activity of the small GTPase RhoA by regulating the level ofArhgap29, a RhoA inactivator. These molecular changes result inincreased formation of actin stress fibers, increased cellular areaand slower migration. This provides a potential molecular

rationale for the observed increased likelihood of post-surgicalcomplications in patients with mutations in IRF6 compared withthose without (Jones et al., 2010).

Our data show delays in wound closure using both an ex vivo

murine embryo culture wound-healing assay and an in vitro

keratinocyte scratch assay. A potential migratory defect of

epithelial cells lacking Irf6 was previously postulated in thezebrafish. Zebrafish embryos injected with a dominant-negativeform of irf6 failed to undergo proper epiboly (Sabel et al., 2009), a

process during which the epithelial enveloping layer moves as acoherent layer to cover the yolk cell. The absence of irf6 in the fishled to the rupture of the embryo at late gastrula stage. Theenveloping layer contains an actin cytoskeleton and cadherins at

the cell–cell junctions (Zalik et al., 1999), reminiscent ofmammalian epithelial cells, suggesting a potential evolutionarilyconserved role for Irf6 in epithelial cell migration.

The defect in keratinocyte migration in the absence of Irf6 isreminiscent of a few murine models. Mice that lack grainy-headlike 3 (Grhl3) are particularly relevant because, like Irf6, Grhl3

encodes a transcription factor that is required to regulate epidermalproliferation and differentiation (Yu et al., 2006). In humans,mutations in IRF6 and GRHL3 have both been identified in VWS(Kondo et al., 2002; Peyrard-Janvid et al., 2014). In addition,

embryos lacking Grhl3 fail to close an ex vivo wound, and Grhl3-deficient keratinocytes were delayed in closing an in vitro scratchwound (Caddy et al., 2010; Hislop et al., 2008). Finally, GRHL3

was identified as a direct target for IRF6 in human keratinocytes(Botti et al., 2011) and in the zebrafish periderm (de la Garza et al.,2013), suggesting that GRHL3 and IRF6 function in a common

pathway in regulating epidermal migration. In support of thishypothesis, keratinocytes that lack either of these genes showedaltered levels of stress fibers and Rho activity. However, whereas

Irf6-deficient keratinocytes display an increase in the prominenceof stress fibers and Rho activity (this study), Grhl3-deficientkeratinocytes display a decrease in stress fiber formation and Rhoactivity (Caddy et al., 2010). Thus, although both genes share a

common function in regulating keratinocyte migration during

wound healing, they appear to act in different pathways thatconverge at regulating the activity of RhoA. Specifically, Irf6 was

shown to regulate Arhgap29 (Leslie et al., 2012), but Grhl3 wasshown to regulate RhoGEF19 (also known as Arhgef19) (Caddyet al., 2010). Future studies will be needed to understand thecomplex gene regulatory network between these two transcription

factors during keratinocyte migration and wound healing.Irf6-deficient keratinocytes were more spread than wild-type cells,

already observable at 1 h after plating and persisting throughout the

course of the scratch-wound assay. Concomitantly, time-lapserecording analysis indicated that Irf6-deficient keratinocytes wereslower cells that traveled less distance. As stress fibers are more

prominent in stationary cells (Couchman and Rees, 1979) and inhibitcell migration (Burridge, 1981), we were not surprised to find thepresence of prominent stress fibers in Irf6-deficient keratinocytes

(1.77 times more cells with prominent stress fibers in the Irf6-deficient group compared with the wild-type group, data not shown),accompanied by an increase in active RhoA. The addition of aROCK inhibitor rescued the migratory phenotype of Irf6-deficient

keratinocytes, thus providing further evidence that the extensivefibers in mutant cells contribute to their slower migration, as reportedpreviously (Arthur and Burridge, 2001). In separate studies, the

addition of the same ROCK inhibitor led to the immortalization ofhuman keratinocytes and increased proliferation (Chapman et al.,2010; McMullan et al., 2003), but this effect was dependent on co-

culture with human fibroblasts (Chapman et al., 2010). Our culturesystem does not contain fibroblasts, and the number of cells dividingduring the scratch assay was not significantly different between the

two groups (data not shown), suggesting that proliferation is unlikelyto contribute to the rescued migratory phenotype.

Irf6 promotes epidermal differentiation, both in vivo and in vitro

(Biggs et al., 2012; Ingraham et al., 2006). If Irf6 is upstream of

RhoA–ROCK, we would hypothesize that increasing levels of RhoAor blocking ROCK would promote epidermal differentiation.However, the keratinocyte-specific RhoA knockout mouse exhibits

a normal epidermis (Jackson et al., 2011). Depletion of ROCKinhibits keratinocyte terminal differentiation in vitro and in vivo (Lockand Hotchin, 2009; Shimizu et al., 2005; Thumkeo et al., 2005), yet it

has no effect on full-thickness wound healing. Interestingly, ROCK-Iand ROCK-II knockout animals exhibit an ‘open-eye’ phenotype – aclassic periderm defect – and an open ventral body wall (Shimizuet al., 2005; Thumkeo et al., 2005). Irf6-deficient mice also exhibit a

mild defect in the ventral body wall (M.D. and B.C.S., data notshown) and a periderm defect (this study; Peyrard-Janvid et al., 2014;Richardson et al., 2009), yet do not exhibit the open-eye phenotype.

Redundancy in the Rho family members, and diversity in targets inthese pathways, is likely to contribute to the observed similarities anddiscrepancies in phenotypes.

Cell–cell and cell–matrix adhesions are crucial components ofcellular migration. Our results show no defect in cell–matrixadhesion at 1 h after plating in the absence of Irf6. This was

rather surprising, based on the increase in both the levels of activeRhoA and the prominence of stress fibers, which have typicallybeen associated with increased cell–matrix adhesion (Arthur andBurridge, 2001), and the increased levels of integrin a2

(Ingraham et al., 2006) and integrin a3 (Botti et al., 2011)reported for Irf6-deficient embryonic skin and adult humankeratinocytes with knockdown of IRF6, respectively. However,

the migratory phenotype was observed during the healing of ascratch wound, which occurs after cells have reached confluenceand therefore have been in culture for at least 48 h. Using

vinculin as an indicator of focal adhesions, we did not detect

Fig. 5. The absence of Irf6 leads to a decrease in the expression ofArhgap29. (A,B) Immunofluorescent staining for Arhgap29 (red) in e17.5embryonic wild-type (A) and Irf6-deficient (B) cutaneous sections.(C) Western blot analyses for Arhgap29 on RIPA extracts from e17.5embryonic skin. (D,E) Immunofluorescent staining for Arhgap29 (red) in wild-type (D) and Irf6-deficient (E) keratinocytes grown on collagen IV in N-medium. Nuclear DNA is labeled with DAPI (blue). Scale bars: 50 mm

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differences in cell–matrix adhesion after 48 h either. None of ourexperiments tested for the strength with which the cells are

attached to the matrix or their ability to sever adhesions from thematrix. Therefore, we cannot rule out the possibility that Irf6-deficient cells are defective in disassembling adhesions, which

would lead to a delay in cellular migration.E-cadherin is a major component of adherens junctions, which

require proper Rho and Rac activity for their establishment and thestabilization of the E-cadherin receptor at the site of intercellular

junction (Braga et al., 1997). Particularly, RhoA signaling isnecessary for cadherin clustering, and its inhibition by p120 cateninaffects nascent cell–cell contacts (Anastasiadis et al., 2000). Although

we have not investigated the role of Irf6 in E-cadherin-mediated cell–cell adhesion, it is interesting to note that altered E-cadherinexpression is observed in the oral epithelium of Irf6-deficient mice

due to a defect in the oral periderm (Richardson et al., 2009).In summary, here, we have identified a novel role for Irf6 in

regulating keratinocyte migration (Fig. 6). Irf6 acts upstream ofArhgap29 to negatively regulate active RhoA. Thus, the loss of Irf6

would lead to an increase in stress fiber formation and slowedmigration. As keratinocyte migration is essential to the processesof wound healing and palatal development, these studies offer a

molecular mechanism for the wound-healing complicationsobserved in patients with VWS. Furthermore, these studies havethe potential to impact on both biological processes and provide

new therapeutic options, as Rho inhibitors are readily available.

MATERIALS AND METHODSMiceAll mice were cared for according to the Animal Care and Use Review

Form at the University of Iowa. Two distinct Irf6 mutant strains were used

interchangeably to obtain Irf6-deficient embryos – Irf6gt1/+ and Irf6del1/+.

Genotyping for the Irf6gt1 allele and the Irf6del1/+ allele was performed as

described previously (Biggs et al., 2012; Ingraham et al., 2006). The

presence of a copulatory plug was designated as embryonic day (e)0.5.

In vivo excisional wound healingTwo 6-mm punch biopsies were performed on the back of 8–12-week-old

wild-type animals, as described previously (Le et al., 2012). Animals (n54–6

per group) were euthanized at 1, 4, 7 and 11 days post-wounding, and wounds

were fixed in 4% paraformaldehyde. Serial sections and immunostaining

were performed as described previously (Biggs et al., 2012; Le et al., 2012).

Ex vivo embryo cultureEmbryonic wound healing was performed as described previously

(McCluskey and Martin, 1995; New and Cockroft, 1979), with

modifications as follows. Embryos were removed from pregnant

females at e10.5. They were dissected out of their amniotic sacs and

left connected to their placentae. To generate the embryonic wound, the

left forelimb bud was amputated using scissors with 4 mm blades. The

embryos were then placed in a conical tube containing 4 ml of filter-

sterilized medium [3 parts 0.9% (w/v) NaCl supplemented with 1%

penicillin-streptomycin, 1 part fresh rat serum]. The vials were gassed at

the beginning of the experiment and every 12 h with 95% O2/5% CO2

and placed in a Bellco Autoblot Micro Hybridization oven at 37 C with

rotation. After 24 h in culture, embryos with a strong heartbeat and good

circulation were processed for scanning electron microscopy. At the end

of the culture, a piece of the tail was removed for genotyping.

Keratinocyte cultureSkin from e17.5 embryos was incubated with 5 U/ml Dispase II (Roche

Diagnostics, Indianapolis, IN) at 4 C for 4 h. The epidermis was peeled

from the dermis and incubated in 0.25% trypsin (Gibco Invitrogen,

Carlsbad, CA) for 20 min at 37 C. Keratinocytes were grown in N-

Medium (Hager et al., 1999), which contains 0.06 mM CaCl2, and were

used after their first or second passage.

In vitro scratch assayScratches were generated with a P200 tip in confluent monolayers of both

wild-type and Irf6-deficient keratinocytes, and static images were

recorded at regular intervals over a 24-h period. Static images were

obtained using a Nikon Eclipse Ti microscope with Nikon Digital Sight

CCD camera and NIS-Elements D 3.0 software (Melville, NY). The open

area was traced using ImageJ. Movies of scratches generated with a P10

tip were captured using a Zeiss Axiovert 200M Mat microscope with

heated and humidified chamber, acquired with a Hamamatsu Orca ER

CCD camera (Bridgewater, NJ) and AxioVision Rel 4.7 software (Zeiss,

Thornwood, NY) and converted to QuickTime format. Analysis of cell

behavior was performed from the QuickTime movies using 2D-DIAS

software, as described elsewhere in detail (Soll and Voss, 1998; Wessels

et al., 2009). Briefly, accurate cell outlines from phase-contrast images

were obtained using the manual trace feature and were converted to beta-

spline replacement images. Total path length, net path length,

instantaneous velocity, persistence and direction change were computed

from the cell centroid position at 10-min intervals over an 18-h period.

Instantaneous velocity was computed by drawing a line from the centroid

in frame n21 to the centroid in frame n+1 and then dividing the length of

that line by twice the time interval between frames (Soll and Voss, 1998).

Directional change was computed as the direction in the interval (n21, n)

minus the direction in the interval (n, n+1). Directional change values

greater than 180˚were subtracted from 360 , resulting in a positive value

between 0˚ and 180 . Persistence was essentially computed by dividing

the speed by direction change, the latter given in grads rather than

degrees, and adding one to the denominator to prevent division by 0.

Area and roundness were computed from contours of the beta-spline

replacement images (Soll and Voss, 1998; Wessels et al., 2004).

Cell-substrate adhesion assayKeratinocytes (passage 2, 21,500 cells/cm2) were plated in 24-well plates

on plastic, collagen-IV- or fibronectin-coated wells (1 mg/cm2, BD

Biosciences, Bedford, MA). Laminin-332-coated plates were prepared

from human keratinocyte cultures. Briefly, confluent keratinocytes were

removed from their matrix by successive incubation first in 1% Triton X-

100 in PBS for 10 min, then in 30 mM Tris-HCl pH 8, 2 M urea in 1 M

NaCl for 10 min and finally in 30 mM Tris-HCl pH 8 in 8 M urea for

10 min. All the buffers contained 0.1% inhibitor cocktail (set III,

Novagen 539134) and 1 mM EDTA. Finally, the coated plates were

washed with 0.1% inhibitor cocktail and 1 mM EDTA in PBS and used

immediately or stored at 280 C (Kirtschig et al., 1995).

After 60 min, the medium was aspirated and the wells were washed

with PBS. Remaining cells were then stained with Giemsa and fixed with

Fig. 6. An Irf6-dependent pathway regulating keratinocytemigration. IRF6 regulates Arhgap29, a GTPase-activating protein thatpromotes the hydrolysis of GTP to GDP, thus returning RhoA to an inactivestate. In the absence of IRF6, Arhgap29 levels are decreased, leading toincreased activity of RhoA, increased prominence of stress fibers andimpaired cellular migration. Blocking the Rho-associated protein kinaseROCK rescues IRF6-dependent migration. IRF6 could also negativelyregulate E-cadherin to promote migration.

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Giordano buffer, as described previously (Biggs et al., 2012). Two

images per well were taken, and the cells were counted and averaged.

Three experiments were performed in duplicate. Similarly, cells were

plated at a density of 21,500 cells/cm2 onto collagen-IV-coated glass

coverslips and fixed in methanol:acetone (75:25, v/v) 60 min later. The

coverslips were then stained with vinculin and phalloidin.

Active RhoA pulldownRho assays were performed as described previously (Ren et al., 1999).

Briefly, the RhoA-binding domain of rhotekin was immobilized on

glutathione-S-transferase-conjugated beads. Cells were lysed in lysis

buffer (50 mM Tris-HCl pH 7.6, 500 mM NaCl, 0.1% SDS, 0.5%

sodium deoxycholate, 1% Triton X-100, 10 mM MgCl2 and 10 mg/ml

each of PMSF, leupeptin and aprotinin), and equal amounts of cell lysate

were incubated with beads with 30 mg of GST-bound rhotekin for 30 min

at 4 C with rotation. Total lysate and bead-conjugated lysate were

prepared for western blotting and run on 10% Bis-Tris gels (Invitrogen).

The membranes were then probed with RhoA-specific antibody. Three

independent experiments were performed. ROCK inhibitor Y27632 was

obtained from Sigma (St Louis, MO) and used at a concentration of

10 mM. Cells were incubated in Y27632 or DMSO control for 24 h and

fixed in 70% ethanol.

AntibodiesMouse monoclonal antibodies against vinculin and Rhodamine-

conjugated phalloidin were obtained from Sigma. Rabbit polyclonal

antibody against Arhgap29 was obtained from Novus Biologicals

(Littleton, CO). Mouse monoclonal antibody against RhoA (clone

26C4) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Anti-mouse-IgG and anti-rabbit-IgG horseradish-peroxidase-conjugated

secondary antibodies were from GE Healthcare (Piscataway, NJ) and

Santa Cruz Biotechnology, respectively.

Protein analysisRadioimmunoprecipitation assay (RIPA) extraction buffer was used for

protein preparation. Equal amounts of protein were separated on 10%

Bis-Tris (Invitrogen) SDS-PAGE gels under denaturing conditions.

Proteins were transferred onto polyvinylidene fluoride membranes (Bio

Rad Laboratories, Hercules, CA), blocked in 10% nonfat dried milk and

incubated with primary antibodies. After incubation with horseradish-

peroxidase-conjugated secondary IgG antibodies, antigen detection was

performed with the chemiluminescent detection system ECL (GE

Healthcare).

MicroscopyKeratinocytes at passage 1 were grown on collagen-IV-coated coverslips

and fixed as described previously (Michel et al., 1996). After blocking

with 3% goat serum (Vector Laboratories, Burlingame, CA), cells were

incubated with primary antibodies, washed in PBS and incubated with

secondary antibodies. 4,6-diamidino-2-phenylindole (DAPI) was used as

a nuclear stain. Images were viewed with a Nikon Eclipse E800

(Melville, NY) and acquired with a SPOT RT slider CCD camera using

Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI).

Black and white images were pseudocolorized and merged. For confocal

microscopy, images were acquired using a Zeiss LSM 710 microscope

(Thornwood, NY) and ZEN 2009 software (Thornwood, NY).

StatisticsData are the means of at least three biological replicates. Statistical

analysis was performed with appropriate tests for each study, as indicated

in the figure legends.

AcknowledgementsThe authors acknowledge Jeff Murray for his unconditional support. The authorsacknowledge the technical assistance of Deborah Wessels at the W.M. KeckDynamic Image Analysis Facility, Katherine Walters at the Central MicroscopyResearch Facility and Lindsey Rhea (University of Iowa, Iowa City, IA), as well aslaboratories from the University of Iowa who provided fresh rat serum. A big thank

you to Paul Martin (University of Bristol, UK) for teaching us the embryonic woundculture technique and Andrew Lidral (University of Iowa) for the use of hismicroscope.

Competing interestsThe authors declare no competing interests.

Author contributionsL.C.B., R.L.N. and M.D. performed experiments; L.C.B., R.L.N., K.A.D., D.R.S.,D.F.L., S.K. and M.D. analyzed the data; L.C.B., K.A.D., B.C.S. and M.D. wrotethe paper.

FundingThis work was partially supported by funding from the National Institutes of Health[grant number AR035313 to M.D. and B.C.S.]; and the National Science Foundation[grant number 1120478 to K.A.D.]. R.L.N. was supported by a grant from the NationalInstitutes of Health for short-term training for students in the health professions [grantnumber 5T35HL007485-34]; D.R.S. was supported by the Developmental StudiesHybridoma Bank at the University of Iowa, a National Resource initiated by theNational Institutes of Health. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139246/-/DC1

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