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,*.

1Department of Pediatrics, The University of Iowa, Iowa City, IA, USA 2Interdisciplinary Graduate Program in Genetics, The University of Iowa, Iowa City, IA, USA 3Department of Biochemistry, The University of Iowa, Iowa City, IA, USA 4Developmental Studies Hybridoma Bank, Department of Biology, The University of Iowa, Iowa City,

IA, USA 5Departments of Microbiology and Molecular Genetics and of Pediatrics and Human Development,

Michigan State University, East Lansing, MI, USA

*Corresponding author:

Martine Dunnwald, PharmD, PhD

Pediatrics

206 MRC

500 Newton Road

The University of Iowa

Iowa City, IA, 52242

319-384-4645

martine-dunnwald@uiowa.edu

Running Title (40 characters): Irf6 and Keratinocyte Migration

Keywords (at least 3 for indexing): Interferon Regulatory Factor 6, migration, keratinocytes

Author contribution: LCB, RLN, and MD performed experiments, LCB, RLN, KAD, DRS, DFL, SK and

MD analyzed the data, LCB, KAD, BCS and MD wrote the paper.

© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 28 April 2014

Summary

Interferon Regulatory Factor 6 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 to wild type. In vitro, Irf6-deficient

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

showed a 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. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and

active RhoA compared to wild type. Blocking ROCK, a downstream effector of RhoA, rescued the

scratch wound delay. Arhgap29, a Rho GTPase Activating Protein was reduced in Irf6-deficient

keratinocytes. Together these data suggest that Irf6 functions through the RhoA pathway to regulate

cellular migration.

Introduction

Cutaneous 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, among which Interferon

Regulatory Factor 6 (Irf6) plays a critical role (Biggs et al., 2012; Botti et al., 2011; Ingraham et al.,

2006).

Interferon Regulatory Factor 6 belongs to the IRF family of transcription factors mediating the

interferon response after viral infection (Honda and Taniguchi, 2006). In contrast to most IRFs, Irf6 is

essential during embryogenesis. Mice lacking Irf6 are perinatal lethal and exhibit limb, craniofacial and

epidermal anomalies (Ingraham et al., 2006; Richardson et al., 2006). In human, mutations in IRF6 cause

Van der Woude and Popliteal Pterygium syndromes, two orofacial clefting disorders (Kondo et al., 2002).

Interestingly, patients with Van der Woude syndrome (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. While Irf6 is expressed in suprabasal

keratinocytes of the epidermis and plays a critical 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.

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Cellular migration is a highly coordinated biological process that includes assembly of cell-cell

and cell-matrix contacts followed by disassembly of older ones (for review (Vicente-Manzanares et al.,

2005)). The coordination of and force required to achieve migration is driven by the reorganization of the

actin cytoskeleton (for review, (Le Clainche and Carlier, 2008)). The actin cytoskeleton normally acts to

provide 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 dissassembles to retract the tail (Le Clainche and

Carlier, 2008; Vicente-Manzanares et al., 2005). Migratory cues are diverse and sensors include clusters

of integrins 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 which subsequently evolve into linear adhesions (Vasioukhin et al., 2000). As the

cytoskeleton requires integrins and cadherins for information about the environment, in turn, contraction

of actin is necessary for assembly of the adhesions that these proteins form with the substrate and other

cells (Vasioukhin et al., 2000).

The Rho family of small GTPases is the central regulator of the 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 support 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 dispensable for epidermal

differentiation but necessary for directed keratinocyte migration (Jackson et al., 2011). RhoA activation is

also required for mediating TGFbeta3-induced stress fibers and TGFbeta3 signaling during palatogenesis

(Kaartinen et al., 2002). Additionally, we identified Arhgap29, a GEF with high affinity for RhoA as a

novel cleft candidate gene downstream of Irf6 (Leslie et al., 2012; Saras et al., 1997). Since Irf6 is

required for proper palatogenesis (Ingraham et al., 2006; Knight et al., 2006) and is a downstream effector

of TGFbeta3 (Knight et al., 2006; Xu et al., 2006), we hypothesize that Irf6 regulates the actin

cytoskeleton in keratinocytes and alters cellular migration.

In this study, we established a role for Irf6 in epithelialization during embryonic wounds. Using

primary keratinocyte culture from wild type and Irf6-deficient embryos in combination with scratch

wound and adhesion assays, we demonstrated that Irf6 is required for proper migration of keratinocytes.

This Irf6-dependent defect is mediated by RhoA.

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Results

Irf6 is present at the wound edge and in the neoformed epidermis following excisional wounds

We used our excisional murine would healing model (Le et al., 2012) to evaluate the expression of

Irf6 in the adult. We observed the presence of Irf6 in keratinocytes of unwounded adult keratinocytes

(Fig. 1A), at the wound edge one day after injury (Fig. 1B) and in cells that just completed

epithelialization (Fig. 1C). Irf6 expression decreased in the neoformed epidermis before returning to

normal level 11 days following injury (data not shown). In the open wound area (Fig. 1B), we noted the

presence of a strong Irf6 signal that likely reflects background staining from necrotic or inflammatory

cells.

Irf6 is required for proper embryonic wound healing.

Irf6-deficient embryos die perinataly from orofacial and epidermal anomalies (Ingraham et al.,

2006), limiting wound healing studies to the embryo. Well-established embryonic wound healing models

have been described that follow epidermal closure after hindlimb resection at e11.5 (McCluskey and

Martin, 1995). Because of the severe hindlimb phenotype in the late stage Irf6-deficient embryos, we

modified 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. 1A,B) and the size of their

wounds immediately following limb removal were identical (Fig.1A-C). Twenty-four hours later, the

wounds of wild type embryos were closed (Fig. 1C,E), while the wounds were significantly open in

embryos deficient for Irf6 (Fig. 1D,E). These data demonstrate that Irf6 is required for proper embryonic

wound healing.

Irf6-deficient keratinocytes are delayed in closing an in vitro scratch wound.

Closure of embryonic wound healing mainly consists of keratinocyte migration across the wound.

To test the hypothesis that Irf6 contributes to epidermal migration, we generated a scratch wound in

confluent monolayers of wild type and Irf6-deficient keratinocytes. Static images were taken at 0-, 6-, 8-,

and 24-hours post scratch (Fig. 2A-F and data not shown). Despite similar initial wound size, Irf6-

deficient keratinocytes were significantly delayed in closing the scratch wound (59% vs 95% at 24h) (Fig.

2E-G).

In order to investigate further the migratory defect of Irf6-deficient keratinocytes, we used time-

lapse video microscopy to generate live imaging of the in vitro scratch assay. Images taken every 5 min

over 18 h revealed that the Irf6-deficient keratinocytes seemed adhered to one another or to the substrate

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whereas wild type keratinocytes moved effortlessly as individual cells (Suppl. Figure 1S). This behavior

can be noted over time in the centroid tracks and stacked perimeter plots generated by 2D-DIAS and

presented in Fig. 2H and I at 10 min intervals. It can be seen from the centroid tracks that the majority of

wild type keratinocytes (Fig. 2H) oriented and moved persistently (small arrows) in the direction of the

scratch wound (large arrow). Perimeter plots revealed that these cells made net progress by the

preferential extension of lamellipodia towards the wound. Irf6-deficient keratinocytes, on the other hand

(Fig. 2I), frequently extended lamellipodia in random directions (small arrows), resulting in reduced

persistent crawling, decreased net progress towards the wound and less persistent tracks. To quantify and

statistically analyze these defects, net path length, total path length and direction change were computed

from centroid positions over time as illustrated in Fig. 2O and described in the Methods section. The net

path (distance from 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 to wild type. In addition, the

average direction change (Fig. 2O, angles , and ) was significantly increased in the absence of Irf6

compared to control (Fig. 2L). Instantaneous velocity of Irf6-deficient keratinocytes was 0.39 m/ hr (±

0.023), approximately half that of wild type cells (0.73 ± 0.054), and the difference was highly significant

(P < 0.001, Fig. 2M). Consequently, persistence of Irf6-deficient keratinocytes was three times lower than

wild type cells (Fig. 2N; P < 0.001). Collectively, our results indicated a deficient directional migration

and reduced speed in cells lacking Irf6, suggesting that Irf6 is necessary for efficient healing of

keratinocyte scratch wounds in vitro.

Irf6-dependent cellular size is independent of the extracellular matrix.

In order to further our understanding of the role of Irf6 in keratinocyte migration, we investigated

cellular adhesion to the substrate one hour after plating. Our data showed no difference in the number of

adhered keratinocytes between wild type and Irf6-deficient keratinocyte (Fig. 3A). We further asked

whether this outcome was dependent on the type of extracellular matrix. Despite an increase in the

number of adherent cells on laminin 332 compared to other substrates (fibronectin, collagen IV and

plastic), we did not detect differences in number of cells adherent between wild type and Irf6-deficient

keratinocytes (Fig. 3B), suggesting that initial cellular adhesion to the extracellular matrix is independent

of Irf6. However, one hour after plating, we consistently observed larger cells in the absence of Irf6

compared to wild type (Fig. 3C vs D). We quantified this observation by measuring the cellular area of

keratinocytes after one hour of plating on plastic, fibronectin, collagen IV and laminin 332. With the

exception of fibronectin, keratinocytes deficient for Irf6 were significantly more spread than their wild

type counterpart (Fig. 3E). Interestingly, cells were more spread when plated on laminin 332 compared to

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any other substrate, but the difference between wild type and mutant was not changed. The plating of Irf6-

deficient keratinocytes on preformed extracellular matrix produced by wild type keratinocytes did not

change the number or the size of adherent Irf6-deficient cells compared to cells plated on collagen IV

(data not shown), supporting the hypothesis that the larger cell size is independent of the extracellular

matrix, but intrinsic to Irf6-deficient keratinocytes.

Focal adhesions are cellular structures involved in cell-matrix adhesion and cellular spreading that

connect the actin cytoskeleton to the point of contact of the cell to the extracellular matrix.

Immunostaining for vinculin, a component of 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, appeared more prominent after one hour of plating in the absence of Irf6. Thus, our

data suggest that Irf6 acts to restrict the spreading of keratinocytes in a substrate-independent fashion by

potentially playing a role on the actin cytoskeleton.

Irf6 regulates the actin cytoskeleton and the amount of active RhoA

Data presented in Figure 3 showed an increase in cellular size after one hour of plating. In cells

grown for a few days in culture, we had previously reported the presence of larger cells in Irf6-deficient

keratinocytes that was not due to epithelial to mesenchymal transition (Biggs et al., 2012). We confirmed

this increase in cellular size in the absence of Irf6 during scratch closure (Fig. 4A) that is accompanied by

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

We first investigated the pattern of actin stress fiber arrangement by staining with phalloidin, a

marker of polymerized actin (Wehland et al., 1977). We observed more prominent stress fibers in the Irf6-

deficient keratinocytes compared to wild type cells (Fig. 4C,D). The percentage of cells with prominent

stress fibers varied from 24.1 to 50.6 across six independent experiments. Combined, Irf6-deficient

keratinocytes had 1.77 times more cells with prominent stress fibers than wild type cells. An increase in

cytoplasmic stress fibers has been previously associated with elevation in RhoA-GTPase leading to an

inhibition of cellular migration (Arthur and Burridge, 2001). In order to test the hypothesis that Irf6

regulates active RhoA, we performed an affinity precipitation assay for GTP-bound RhoA, the active

form of RhoA (Fig. 4E). We observed an increase in active RhoA in Irf6-deficient keratinocytes

compared to wild type cells. These results confirm a role for Irf6 in negatively regulating stress fibers via

RhoA.

To determine whether Irf6-dependent stress fibers and scratch wound healing delay was 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 were treated with Y27632. Irf6-deficient

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keratinocyte cultures exhibited a greater reduction of stress fibers, yet both cultures had reduced actin

stress fibers (Fig. 4F, G). These data suggest that blocking ROCK partially rescued the phenotypic

characteristics of Irf6-deficient keratinocytes, furthering our hypothesis that Irf6 regulates RhoA. To test

whether this partial rescue of stress fibers had functional consequences, we scratched wild type and Irf6-

deficient confluent keratinocytes in the presence of Y27632. After 18 h, both wild type and Irf6-deficient

scratches were 80% closed without statistical significant difference between the groups (Fig. 4H).

Together, these data indicate that Irf6 regulates the balance of active and inactive RhoA, thus controlling

stress fiber formation, and keratinocyte migration.

In order to investigate further the effect of Y27632, we analyzed time-lapse video microscopy of

wild type and Irf6-deficient keratinocytes treated with the ROCK inhibitor as described for Figure 2. Our

results show 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. 4K), and consequently the persistence (Fig. 4L) of Irf6-deficient keratinocytes. Collectively,

our results indicated a rescue of deficient directional migration and reduced speed in cells lacking Irf6

with ROCK inhibitor, but not cellular size and shape.

Irf6 regulates Arhgap29 to modulate RhoA activity

Two classes of proteins regulate RhoA activity: GAPs and GEFs, inactivating and activating

RhoA, respectively (Cherfils and Zeghouf, 2013). To identify how Irf6 regulates RhoA levels, we

searched our microarray data comparing wild type to Irf6-deficient embryonic skin for either decreased

GAPs or increased GEFs (Ingraham et al., 2006). We identified Arhgap29 as a candidate because it was

expressed at higher levels in the skin than the other GAPs, and there was a non-significant, but reduced

expression in the absence of Irf6. We confirmed the presence and the decrease in Arhgap29 levels at the

protein level in cutaneous tissues of e17.5 Irf6-deficient embryos compared to wild type by

immunostaining (Fig. 5A,B) and western blot analysis (Fig. 5C) and. In vivo, Arhgap29 was mainly

expressed throughout the epidermis, with some 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 protein appeared perinuclear within the cytoplasm of the cells in a punctate pattern, with no apparent

alteration of localization between wild type and Irf6-deficient keratinocytes. However, levels of Arhgap29

expression were reduced in the absence of Irf6 (Fig. 5D, E). These data provide evidence that Arhgap29, a

key regulator of RhoA activity, lies downstream of Irf6 in keratinocytes.

Discussion

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Using our Irf6-deficient murine model (Biggs et al., 2012; Ingraham et al., 2006), we demonstrate

that Irf6 acts as a regulator of keratinocyte migration. We show that Irf6 inhibits the activity of the small

GTPase RhoA by regulating the level of Arhgap29, a RhoA inactivator. These molecular changes result in

increased actin stress fibers, larger cellular area as well as slower migration. This provides a potential

molecular rationale for the observed increased likelihood of post-surgical complications observed in

patients with mutations in IRF6 compared to those 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 the zebrafish. Zebrafish embryos injected with a dominant-

negative form of irf6 failed to undergo proper epiboly (Sabel et al., 2009), a process during which the

epithelial enveloping layer moves as a coherent layer to cover the yolk cell. The absence of irf6 in the fish

led to the rupture of the embryo at late gastrula stage. The enveloping layer contains an actin cytoskeleton

and cadherins at the cell-cell junctions (Zalik et al., 1999), reminiscent of mammalian epithelial cells,

suggesting a potential evolutionarily conserved role for Irf6 in epithelial migration.

The defect in keratinocyte migration in the absence of Irf6 is reminiscent of a few murine models.

Mice that lack grainy-head like 3 (Grhl3) are particularly relevant because, like Irf6, Grhl3 encodes a

transcription factor that is required to regulate epidermal proliferation and differentiation (Yu et al.,

2006). In human, mutations in IRF6 and GRHL3 have both been identified in Van der Woude syndrome

(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 scratch wound (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

these two genes function in a common pathway in regulating epidermal migration. In support of this

hypothesis, keratinocytes that lack either of these genes showed altered levels of stress fibers and Rho

activity. However, whereas Irf6-deficient keratinocytes have an increase in stress fibers and Rho activity

(this study), Grhl3-deficient keratinocytes have a decrease in stress fibers and Rho activity (Caddy et al.,

2010). Thus, while both genes share a common function of regulating keratinocyte migration during

wound healing, they appear to act in different pathways that converge at regulating the activity of RhoA.

Specifically, Irf6 was shown to regulate Ahrgap29 (Leslie et al., 2012), but Grhl3 was shown to regulate

RhoGEF19 (Caddy et al., 2010). Future studies will be needed to understand the complex 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 from one hour after

plating and throughout the course of the scratch wound. Concomitantly, time-lapse recording analysis

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indicated slower cells that travelled less distance. As stress fibers are more prominent in stationary cells

(Couchman and Rees, 1979) and inhibit cell migration (Burridge, 1981), we were not surprised to find the

presence of prominant stress fibers in Irf6-deficient keratinocytes (1.77 fold more cells with prominent

stress fibers in Irf6-deficient group compared to wild type, data not shown), accompanied by an increase

in active RhoA. The addition of a ROCK inhibitor rescued the migratory phenotype of Irf6-deficient

keratinocytes, thus providing further evidence that the extensive fibers in mutant cells contribute to their

slow migration as previously reported (Arthur and Burridge, 2001). In separate studies, the addition of the

same ROCK inhibitor led to the immortalization of human keratinocytes and increased proliferation

(Chapman et al., 2010; McMullan et al., 2003), yet this effect was dependent on co-culture with human

fibroblasts (Chapman et al., 2010). Our culture system does not contain fibroblasts, and the number of

cells dividing during the scratch assay was not significantly different between the two groups (data not

shown), suggesting that proliferation is unlikely to 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 RhoA or blocking

ROCK would promote epidermal differentiation. However, the keratinocyte specific RhoA knockout

mouse exhibited a normal epidermis (Jackson et al., 2011). Depletion of ROCK inhibited keratinocyte

terminal differentiation in vitro and in vivo (Lock and Hotchin, 2009; Shimizu et al., 2005; Thumkeo et

al., 2005), yet it had no effect on full thickness wound healing. Interestingly, ROCK-I and –II knockout

animals exhibited an open eyes phenotype – a classic periderm defect – and an open ventral body wall

(Shimizu et al., 2005; Thumkeo et al., 2005). Irf6-deficient mice also exhibit a mild ventral body wall

defect (Dunnwald and Schutte, personal communication) and periderm defect (this study and (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 in these pathways is likely to contribute to the observed

similarities and discrepancies in phenotypes.

Cell-cell and cell-matrix adhesions are critical components of cellular migration. Our results show

no defect in cell-matrix adhesion after one hour of plating in the absence of Irf6. This was rather

surprising, based on the increase in active RhoA and stress fibers that have typically been associated with

increased cell-matrix adhesion (Arthur and Burridge, 2001) and the increased levels of integrin alpha 2

(Ingraham et al., 2006) and integrin alpha 3 (Botti et al., 2011) reported in Irf6-deficient embryonic skin

and adult human keratinocytes knockdown for IRF6, respectively. However, the migratory phenotype was

observed during a scratch wound, which is performed after cells have reached confluence and therefore

have been in culture for at least 48 h. Using vinculin as an indicator of focal adhesions, we did not detect

differences in cell-matrix adhesion after 48 h either. None of our experiments tested for the strength with

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which the cells are attached to the matrix or their ability to sever adhesions from the matrix. Therefore, we

cannot rule out the possibility that Irf6-deficient cells are defective in disassembling adhesions properly,

which would lead to a delay in cellular migration.

E-cadherin is a major component of adherens junctions that require proper Rho and Rac activity

for their establishment and the stabilization of the E-cadherin receptor at the site of intercellular junction

(Braga et al., 1997). Particularly, RhoA signaling is necessary for cadherin clustering and its inhibition by

p120 catenin affects nascent cell-cell contact (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 altered E-

cadherin expression in the oral epithelium of Irf6-deficient mice due to a defect in the oral periderm

(Richardson et al., 2009).

In summary, we identified a novel role for Irf6 in regulating keratinocyte migration (Fig. 6). Irf6

acts upstream of Arhgap29 to negatively regulate active RhoA. Thus, the loss of Irf6 would lead to an

increase in stress fibers and slow migration. As keratinocyte migration is essential to wound healing and

palatal development, these studies offer a molecular mechanism for the wound healing complications

observed in patients with VWS. Furthermore, these studies have the potential to impact both biological

processes, and provide new therapeutic options, as Rho inhibitors are readily available.

Materials and Methods

Mice

All mice were cared for according to the ACURF at the University of Iowa. Two distinct Irf6

mutant strains were used interchangeably to obtain Irf6-/- embryos, Irf6gt1/+ and Irf6del1/+. Genotyping for

the Irf6gt1 allele and the Irf6del1/+ allele was performed as described (Biggs et al., 2012; Ingraham et al.,

2006). The presence of a copulatory plug is designated as embryonic (e) 0.5.

In vivo excisional wound healing

Two 6-mm punch biopsies were performed on the back of 8-12 weeks old wild type animals as

previously described (Le et al., 2012). Animals (N = 4-6 per group) were euthanized 1, 4, 7 and 11 days

post-wounding and wounds fixed in 4% paraformaldehyde. Serial sections and immunostaining were

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

Ex vivo embryo culture

Embryonic wound healing was performed as described (McCluskey and Martin, 1995; New and

Cockroft, 1979) with modifications as follows. Embryos were removed from pregnant females at e10.5.

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They were dissected out of their amniotic sacs and left connected to their placenta. 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 media (3 parts 0.9% 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 37ºC 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 culture

Skin from e17.5 embryos was incubated with 5 U/ml Dispase II (Roche Diagnostics, Indianapolis,

IN) at 4ºC for 4 h. The epidermis was peeled from the dermis and incubated in 0.25% trypsin (Gibco

Invitrogen, Carlsbad, CA) for 20 min at 37ºC. Keratinocytes were grown in NMedium (Hager et al.,

1999) which contains 0.06 mM CaCl2, and used after their first or second passage.

In vitro scratch assay

Scratches were generated with a P200 tip in confluent monolayers of both wild type and Irf6-/-

keratinocytes and static images 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 (http://rsbweb.nih.gov/ij/). Movies

of scratches generated with a P10 tip were captured using a Zeiss Axiovert 200M Mat microscope with

heated and humidified chamber and acquired with 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 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 n-1 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 (n-1, 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

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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 assay

Keratinocytes (passage 2, 21,500 cells/cm2) were plated in 24-well plates on plastic, collagen IV-

or fibronectin-coated wells (1 g/cm2 BD Biosciences, Bedford, MA). Laminin 332-coated plates were

prepared from human keratinocytes cultures. Briefly, confluent keratinocytes were removed from their

matrix by successive incubation in 1 % Triton X-100 in PBS, 30 mM Tris pH 8 and 2 M urea in 1M

NaCl, and 30 mM Tris pH 8 in 8 M urea for 10 min in each. 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 immediately used or stored at -80C (Kirtschig et al.,

1995).

After 60 min, the media was aspirated and the wells washed with PBS. Remaining cells were then

stained with Giemsa and fixed with Giordano buffer, as previously described (Biggs et al., 2012). Two

images per well were taken, and cells counted and averaged. Three experiments were performed in

duplicate. Similarly, 21,500 cells/cm2 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 Pulldown

Rho assays were completed as described (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, pH 7.6, 500 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mM

MgCl2, and 10 g/ml of each PMSF, leupeptin, and aprotinin) and equal amount of protein was incubated

with beads with 30 µg og protein for 30 min at 4ºC 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 antibody. Three independent experiments were performed.

ROCK inhibitor Y27632 was obtained from Sigma (St. Louis, MO) and used at a concentration of

10 M. Cells were incubated in Y27632 or DMSO control for 24 h and fixed in 70% ethanol.

Antibodies

Mouse monoclonal antibodies against vinculin and rhodamine-conjugated phalloidin were

obtained from Sigma (St. Louis, MO). Rabbit polyclonal antibody against Arhgap29 was obtained from

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Novus Biologicals (Littleton, CO). Mouse monoclonal antibody against RhoA (clone 26C4) was obtained

from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse and anti-rabbit horseradish

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

Biotechnology, Inc., respectively.

Protein analysis

Radioimmunoprecipitation assay 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 (BioRad Laboratories,

Hercules, CA), blocked in 10% nonfat dry milk and incubated in primary antibodies. After incubation

with horseradish peroxidase-conjugated secondary IgG antibodies, antigen detection was performed with

the chemiluminescent detection system ECL (GE Healthcare).

Microscopy

Keratinocytes in passage 1 were grown on collagen IV-coated coverslips and fixed as described

(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 in 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).

Statistics

Data are averages of at least three biological replicates. Statistical analysis was performed with

appropriate tests for each study as indicated in the figure legend.

Funding

This work was partially supported by funding from the National Institute of Health (AR035313 to

M.D., B.C.S.) and National Science Foundation (1120478 to K.A.D). R. N. was supported by a grant for

short-term training for students in the health professions 5T35HL007485-34. D.R.S was supported by the

Developmental Studies Hybridoma Bank at the University of Iowa, a National Resource initiated by the

National Institute of Health.

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Acknowledgements

The authors acknowledge Jeff Murray for his unconditional support. The authors acknowledge the

technical assistance of Deborah Wessels at the W.M.Keck Dynamic Image Analysis Facility, Katherine

Walters at the Central Microscopy Research Facility, and Lindsey Rhea, as well as laboratories from the

University of Iowa who provided fresh rat serum. A big thank you to Paul Martin (University of Bristol)

for teaching us the embryonic wound culture technique, and Andrew Lidral (University of Iowa) for the

use of his microscope.

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Figure legends

Figure 1: Irf6 is expressed in adult wound healing and required for embryonic wound healing.

Immunofluoresent staining for Irf6 (red) and nuclear DNA (DAPI, blue) of adult back skin (A), excisional

wounds one day (B; white arrow head indicates the wound edge) and seven days (C) post injury.

Scanning electron microscopic images of wild type (D, G) and Irf6-deficient (E, H) E10.5 embryos.

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Forelimbs were sectioned to create a wound (white arrow). The size of the original wounds was identical

between wild type and Irf6-deficient animals (F). After twenty-four hours, wild type wounds were largely

closed (G, I) while wounds from Irf6-deficient embryos remain open (H, I). *P < 0.05. hl = hindlimb.

Scale bar for A-C = 50 µm; scale bar for C, D = 1 mm; scale bar for G, H = 100 µm.

Figure 2: Impaired closing of a scratch wound because of reduced speed and directionality in Irf6-

deficient keratinocytes.

(A-G) Still recording of in vitro scratch wounds of wild type (A, C, E) and Irf6-deficient (B, D, F)

keratinocytes. Cells were grown to confluency (A, B), then scratched with a yellow tip (C, D). Twenty-

four hours later, wild type keratinocytes closed the scratch (E) while Irf6-deficient cells did not (F).

Quantification of percentage of open area over time (G). (H, J) 2D-DIAS generated centroid tracks and

stacked perimeter plots of representative wild type (H) and Irf6-deficient (I) keratinocytes. Large arrow at

the bottom of each panel indicates the direction of the scratch wound and small arrows indicate cellular

direction of travel. The final cell perimeter in each perimeter plot is color-coded gray. (J - N) Analysis of

live video recording of in vitro scratch wounds. (O) Method for calculating net path length, total path

length and direction change. *P < 0.05; ***P < 0.001 after Student t-test. Scale bar = 100 µm

Figure 3: Irf6-dependent cellular size is independent of the 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 one hour later. Distribution of cells per microscopic field (A) or average of

number of cells on different extracellular matrices (B). Confocal images of vinculin (green) and

phalloidin (red) staining of wild type (C) and Irf6-deficient keratinocytes (D). Nuclear DNA is labeled

with DAPI (blue). Area of wild type and Irf6-deficient keratinocytes was measured after an hour of

plating on different extracellular matrices (E). Number of cells analyzed varied from 40 (Irf6-deficient

keratinocytes on fibronectin) to 1211 (Irf6-deficient keratinocytes on Lam). ***P < 0.001; NS = non

significant after Student t-test. Scale bar = 20 µm

Figure 4: Irf6-dependent changes in actin cytoskeleton leads to increase active RhoA. Area (A) and

roundness (B) of wild type and Irf6-deficient keratinocytes were analyzed during the scratch wound assay

of cells grown with or without Y27632 (Y) on collagen IV. Confocal images of vinculin (green) and

phalloidin (red) staining of wild type (C, F) and Irf6-deficient keratinocytes (D, G) grown without (C, D)

or with (F, G) Y27632. Nuclear DNA is labeled with DAPI (blue). (E) Affinity precipitation assay for

GTP-bound RhoA on wild type and Irf6-/- keratinocytes probed for RhoA. (H) Percentage of scratch

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closure of wild type and Irf6-deficient keratinocytes grown with or without Y27632 (N = 3-5). (I - L)

Analysis of live video recording of in vitro scratch wounds. White arrow heads: cortical actin; white

arrows: stress fibers. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 after One-way Anova. Scale

bar = 20 µm. Note that data for samples without Y27632 are the same as the one presented in figure 2.

Figure 5: Absence of Irf6 leads to decrease in Arhgap29. Immunofluoresence for Arhgap29 (red) on e17.5

embryonic wild type (A) and Irf6-deficient (B) cutaneous sections. Western blot analyses for Arhgap29

(C) on e17.5 embryonic skin RIPA extracts. Immunofluorescence for Arhgap29 (red) of 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 µm

Figure 6: Irf6-dependent pathway regulating keratinocyte migration

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