GEF-H1 functions in apical constriction and cell intercalations and is essential for vertebrate neural

36 Pages·2014·3.51 MB·English

(cid:2)2014.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2014)127,2542–2553doi:10.1242/jcs.146811
RESEARCH ARTICLE
GEF-H1 functions in apical constriction and cell intercalations and
is essential for vertebrate neural tube closure
Keiji Itoh*, Olga Ossipova* and Sergei Y. Sokol`
ABSTRACT 2013). Taken together, these cell behaviors are the main
contributors to morphogenetic movements underlying vertebrate
Rho family GTPases regulate many morphogenetic processes
neurulation.
duringvertebrate developmentincludingneuraltubeclosure. Here
One of the crucial regulators of neural tube closure is non-
we report a function for GEF-H1/Lfc/ArhGEF2, a RhoA-specific
muscle myosin II (Rolo et al., 2009; Tullio et al., 2001).
guanine nucleotide exchange factor that functions in neurulation
Both intercalatory and constricting cell behaviors require
in Xenopus embryos. Morpholino-mediated depletion of GEF-H1 reorganization of many cytoskeletal components and involve
resulted in severe neural tube defects, which were rescued by dynamic changes in myosin II localization and activity (Lecuit
GEF-H1 RNA. Lineage tracing of GEF-H1 morphants at different etal.,2011;Rauzietal.,2010;Vicente-Manzanaresetal.,2009).
developmental stages revealed abnormal cell intercalation and MyosinIIcontrolsthecontractileforcebybindingto,andsliding
apical constriction, suggesting that GEF-H1 regulates these cell along, actin filaments. Myosin II has also been implicated in
behaviors. Molecular marker analysis documented defects in a variety of cellular processes – such as cell junction stability,
myosin II light chain (MLC) phosphorylation, Rab11 and F-actin nuclear movement, apical–basal protein targeting – yet there is
accumulationinGEF-H1-depletedcells.Ingain-of-functionstudies, limitedunderstandingofhowmyosinIIisselectivelyrecruitedto
overexpressedGEF-H1inducedRho-associatedkinase-dependent perform these specific tasks. Similar to myosin II, small Rho
ectopic apical constriction – marked by apical accumulation of GTPases play essential roles in vertebrate neurulation (Habas
phosphorylatedMLC,c-tubulinandF-actininsuperficialectoderm– etal.,2003;Habasetal.,2001;Hall,2005;JaffeandHall,2005;
and stimulated apical protrusive activity of deep ectoderm cells. Kinoshita et al., 2008). The existence of at least 70 vertebrate
guanine nucleotide exchange factors (GEFs) that are related to
Takentogether,ourobservationsnewlyidentifyfunctionsofGEF-H1
Dbl(alsoknownasMCF2),whichareactivatorsofRhoGTPases,
inmorphogeneticmovementsthatleadtoneuraltubeclosure.
with unique spatiotemporal expression, subcellular localization
KEYWORDS:Neuraltubeclosure,Apicalconstriction,Epiboly, and diverse specificities presents a significant challenge for
Cellintercalation,Xenopus,Lfc,MyosinII,RhoGTPases, studies of the regulation of Rho GTPases in the context of the
Morphogenesis whole organism (Rossman et al., 2005).
Only a few of the existing RhoA-specific GEFs have been
assigned specific developmental functions. The mesoderm-
INTRODUCTION specific WGEF (also known as ARHGEF19) (Tanegashima
Although neural tube defects are among the most common et al., 2008) and ubiquitous Net1 (Miyakoshi et al., 2004) have
developmentalabnormalities,andmorethan200geneshavebeen been implicated in convergent extension movements. The Rho
implicated in this process in mouse genetic models (Harris and GEF Trio functions in neural crest migration (Kashef et al.,
Juriloff, 2010), the molecular and cellular events that regulate 2009) and apical constriction during optic lens pit invagination
neural tube closure are poorly understood. The development of (Plagemanetal.,2011).InDrosophila,RhoGEF2isessentialfor
the vertebrate central nervous system is a complex process that the apical constriction that is associated with tissue invagination
involves a number of distinct steps. Initially, neural tissue is during gastrulation (Barrett et al., 1997; Ha¨cker and Perrimon,
specifiedindorsalectodermbysecretedfactorsthatareproduced 1998; Ko¨lsch et al., 2007). Although vertebrate neurulation is
by the Spemann organizer (De Robertis and Kuroda, 2004). likely to involve many GEFs, at present, only PDZ-RhoGEF/
During gastrulation, neuroectoderm progenitors exhibit complex ArhGEF11 has been implicated in this process in the chicken
morphogenetic behaviors to conform to the main embryonic (Nishimura et al., 2012). In zebrafish embryos, ArhGEF11
body plan (Gray et al., 2011; Keller et al., 1992a; Poznanski morphants do not exhibit defects in neural tube morphogenesis
et al., 1997). As development proceeds, apical constriction of (Panizzietal.,2007),raisingaquestionastowhetherthisprotein
neuroepithelial cells, cell intercalations and dorsal convergence has a conserved role in neural tube closure. In-depth analysis
e
drive the appearance of bilateral neural folds and their fusion of individual GEF proteins is therefore essential for further c
n
(ColasandSchoenwolf,2001;DavidsonandKeller,1999;Keller, understanding of the morphogenetic mechanisms that underlie e
1991; Schroeder, 1970; Suzuki et al., 2012; Wallingford et al., vertebrate neurulation. Sci
GEF-H1/Lfc/ArhGEF2 is a microtubule-binding Dbl- ell
DepartmentofDevelopmentalandRegenerativeBiology,IcahnSchoolof homology-domain GEF that is specific for RhoA (Birkenfeld C
MedicineatMountSinai,NewYork,NY10029,USA. etal.,2008;Glavenetal.,1999;Krendeletal.,2002;Renetal., f
o
*Theseauthorscontributedequallytothiswork 1998).Inculturedmammaliancells,GEF-H1hasbeenimplicated
al
`Authorforcorrespondence([email protected]) in cell adhesion (Birukova et al., 2006; Chang et al., 2008) n
r
(Yamahashi et al., 2011), cell migration (Heasman et al., 2010; u
o
Received3December2013;Accepted16March2014 Nalbantetal.,2009;Tsaparaetal.,2010),cytokinesis(Birkenfeld J
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RESEARCHARTICLE JournalofCellScience(2014)127,2542–2553doi:10.1242/jcs.146811
etal.,2007),junctionalcommunication(Aijazetal.,2005;Benais- of GEF-H1 efficiently induces Rho-associated kinase (ROCK)–
Pontetal.,2003;Guillemotetal.,2008)andvesiculartrafficking myosin-II-dependent apical constriction in embryonic ectoderm
(Pathak et al., 2012). Despite these crucial roles of GEF-H1 in and stimulates the protrusive activity of deep ectoderm cells.
regulating cell shape and behavior in vitro, its involvement These observations reveal an important role for GEF-H1 in
in morphogenetic processes in vivo has not been investigated. morphogenetic events that accompany vertebrate neural tube
Because GEF-H1 RNA is expressed in the early ectoderm, and closure.
the neural plate, of Xenopus embryos (Morgan et al., 1999), we
investigated a possible role for GEF-H1 in neurulation. In this RESULTS
study, we demonstrate that depletion of GEF-H1 from Xenopus DepletionofXenopusGEF-H1resultsinneuraltube
embryosinterfereswithmorphogeneticcellmovements,including closuredefects
cell intercalation and apical constriction, thereby leading to GEF-H1 RNA is expressed in the neural tissue of Xenopus
neurulation defects. In gain-of-function studies, overexpression embryos (Morgan et al., 1999), indicating a possible function
Fig.1. NeuraltubedefectsinembryosdepletedofGEF-H1.(A)Uninjectedembryoatstage16.(B–D)Four-cellembryoswereunilaterallyinjectedwith
control(CoMO)(B),orGEF-H1a(GEF-H1aMO)(C,D)morpholinooligonucleotides(20ngeach)andLacZRNA(40pg).(D)HumanGEF-H1RNA(60pg)
rescuedtheneuralfolddefectthatwascausedbyGEF-H1aMO.Thedorsalviewisshown,anterioristothetop.Arrowspointtotheneuralfolds.(E,F)Neural e
c
foldmorphologyinstage-17(st17)embryosthathadbeeninjectedwithCoMO(40ng)(E)orGEF-H1MO(F)(40ng)wasvisualizedbyusingb-catenin n
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staining(green).Membrane-associatedRFPRNA(160pg)(F)markstheinjectedsideoftheembryo.N,notochord;S,somite.Tissueboundariesare ci
demarcatedbybrokenlines.Arrowheadpointstoconstrictingcells,*lackofapicalconstriction.(G,H)Constrictedcellmorphologyinthecross-sectionsof S
cnheaunrunlealeomnlbyryoofsH(.sTtahgeeb1ro5k)ethnaltinheasdinbeGenanudnilHa9tesrhaollywinthjeectceedllwboithunGdEarFie-Hs.1MM,Omi(d4l0inengp)o.s(Giti)onco,ndtororsla(lcois)aetmthberytoo.p(.HA)rGroFwPs(pgoreinetnt)oiscoanlsintreicatginegtrcaecllesr,,*(Hla9c)kreodf ell
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apicalconstriction.(I)QuantificationofneuralfolddefectscausedbytheinjectionofGEF-H1aMOandtherescuewithhumanorXenopusGEF-H1RNA.
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MorpholinooligonucleotidesandRNAswereinjectedattheindicateddoses.Neuraltubedefectswerescoredatstages16–18asdescribedinsupplementary o

materialFig.S1.Thenumberofembryosthatwerescoredisshownalongthetop.(J)QuantificationofcellshapeinsuperficialneuroectodermfromGEF-H1- al
MO-injectedorcontroluninjectedsides.Resultsareexpressedastheratiosofapical-basallengthovertheapicalwidth(ABL/AW)infourtosixsuperficial n
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cellsthatwerelocatedadjacenttothemidline.Means6s.d.areshown.Cellswerescoredin8–11sectionsfromthreeembryos,withthetotalcellnumbershown u
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ontopofeachgroup.StatisticalsignificancewasassessedbyusingStudent’st-test,P,0.0001.Scalebars:100mm(E);20mm(G). J
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RESEARCHARTICLE JournalofCellScience(2014)127,2542–2553doi:10.1242/jcs.146811
duringneuraltubeformation.Tostudythedevelopmentalrolesof (Fig. 1F–H; supplementary material Fig. S1A,C). Examination
GEF-H1, we depleted the Xenopus GEF-H1 protein with of the superficial cell morphology confirmed a defect in apical
antisense morpholino oligonucleotides. Because there are two constriction, an important cell behavior that accompanies neural
Xenopus laevis GEF-H1 cDNAs (a and b, see Materials and tube closure (Fig. 1G,H,J).
Methods), which differ in their 59-untranslated sequence and To confirm neural tube closure defects in GEF-H1-MO-
several N-terminal amino acids, we used morpholino injected embryos using a different assay, we visualized the
oligonucleotides that targeted different sequences. Using the neural plate boundary by whole-mount in situ hybridization for
morpholino oligonucleotides GEFH1 MO and GEF-H1a MO, Sox2(apan-neural-tissuemarker;Fig. 2).Sox2wasmorebroadly
which both targeted the GEF-H1a isoform, we observed dose- distributed and less intensely stained at the side of the embryo
dependent neural fold defects in embryos that had been thathadbeeninjectedwitheitherofthetwoGEF-H1morpholino
unilaterally injected with both morpholino oligonucleotides oligonucleotides compared with the control side (Fig. 2A,D,E),
compared with the control siblings (Fig. 1A–C,F; which is consistent with the observed neural folding defects. All
supplementary material Fig. S1A and Movie 1). Similar neural three morpholino oligonucleotides produced similar defects on
tube defects were seen upon using the morpholino the width of the Sox2 expression domain (Fig. 2A,D,E; data not
oligonucleotide GEF-H1b MO, which targets a different protein shown). This defect was rescued by wild-type human GEF-H1
isoform and synergized with the GEF-H1a MO in co-injection but not by the inactive form of human GEF-H1, which has the
experiments (supplementary material Fig. S1A). GEF-H1a MO mutation Y393A (Fig. 2A–C,F), further suggesting that GEF-H1
and GEF-H1 MO efficiently inhibited Xenopus GEF-H1a RNA activity is required for the morphogenetic behaviors that
translation in vivo (supplementary material Fig. S1B), and both accompany neural fold formation. Taken together, these results
wereusedinsubsequentloss-of-functionstudies.Importantly,co- revealanessentialroleofGEF-H1incellshapechangesthatare
injection of human GEF-H1 or Xenopus GEF-H1 RNAs lacking associated with neural tube closure.
the morpholino targetsequence significantly rescued neural tube
defects in GEF-H1-MO-injected embryos (Fig. 1D,I). GEF-H1regulatesneuraltubeclosurebyaffectingdifferent
Weexaminedcellandtissuemorphologyinstage-17neurulae cellbehaviors
thathadbeenunilaterallyinjectedatthe4–8-cellstagewithGEF- InordertounderstandhowGEF-H1depletionleadstoneuraltube
H1 MO and membrane-associated red or green fluorescent closure defects, we wanted to examine specific cell behaviors in
protein (RFP and GFP, respectively) RNAs as lineage tracers. GEF-H1-depleted tissues at different developmental stages.
Cryosections demonstrated that the neural fold failed to close at Besides the altered cell shape in the closing neural tube in GEF-
the GEF-H1-MO- or GEF-H1a-MO-injected side compared H1 morphants, we studied the effect of GEF-H1 morpholino
with the uninjected side or control-MO-injected embryos oligonucleotides on apical constriction using several markers of
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Fig.2. ChangesintheSox2-positivedomaininGEF-H1morphants.(A–E)Embryoswereinjectedlaterallyintotwositesatthefour-cellstagewithLacZ S
R(2N0Anga)s,(liEn)eaGgEeFt-rHac1eMr(O40(4p0g)nga)ndanmdohrupmhoalinnoGoElFig-oHn1ucRleNoAtid(GesEFor-HR1N;Awst)a(s60inpdgic)aoterdh.uDmoasnesGwEFer-eH1a-sYf3o9ll3oAwsR:N(AA–(CY)3G93EAF)-H(810a0MpOg).aWndho(Dle)-mCoouMnOtin(cosintutrol) ell
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hybridizationwithaSox2probeatstages14–15isshown,b-galactosidaseactivity(red)markstheinjectedside.Adorsalviewisshown,theanterioristop.
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(B)Wild-typehumanGEF-H1butnotthe(C)inactiveGEF-H1-Y393ARNAreversesthemorphantphenotype(A).(A–E)Whitebarsindicatethewidthofthe o

Sox2expressiondomainatthelevelofthemid-hindbrainboundary.(F)Quantificationofembryophenotypes.TheSox2domainwasmeasuredatthemid- al
hindbrainboundaryasindicatedinA–D.TheratiosoftheSox2domainwidthontheinjectedsideoverthatontheuninjectedsideareshown.Aratioof1.0 n
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indicatesequalwidthoftheSox2domainonbothsides.StatisticalsignificancewasassessedbyusingStudent’st-test.***P,0.0001.Thenvaluesareshown u
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acrossthetopofthegraphandindicatethenumberofembryosthatweremeasured. J
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apicalconstriction(Fig. 3).Weobservedthatthesuperficialneural that GEF-H1 regulates cell behaviors in both neural and non-
plate cells on the side that had been injected with GEF-H1 MO neural tissues.
failed to express phosphorylated regulatory myosin II light Epiboly is an early morphogenetic process that is driven by
chain (MLC) and F-actin, known molecular markers of apical radial cell intercalations, which convert multilayered blastula
constriction (Choi and Sokol, 2009; Lee and Harland, 2007) ectoderm into two cell-layers that eventually spread over the
(Fig. 3A–D).Additionally,thesuperficialcellsdidnotaccumulate entire embryo surface during gastrulation (Keller, 1991). To
Rab11,a recycling endosomemarkerthat has been implicated in assess the involvement of GEF-H1 in epiboly, we studied clone
neuraltubeclosure(Ossipovaetal.,2014)(Fig. 3E,F).Bycontrast, spreadinginstage-11embryosthathadbeenunilaterallyinjected
thesemarkersandtissuemorphologywerenotaffectedbyinjection with GEF-H1 morpholino oligonucleotides at the four- to eight-
of the control MO (supplementary material Fig. S1D–F). Taken cell stage and then injected bilaterally with LacZ RNA at the
together with the previously shown cell morphology data 16-cell stage (Fig. 4D). The side that received either of the two
(Fig. 1G,H,J), these findings further support the conclusion that GEF-H1 morpholino oligonucleotides consistently showed more
theapicalconstrictionofneuralplatecellsisabnormalinGEF-H1 restricted distribution of b- galactosidase-positive cells in 30 out
morphants. of the 35 examined embryos by contrast with that of the control
In addition to neural tube closure defects, we observed that side. This was confirmed by measuring the b-galactosidase-
non-neural ectoderm contains three to seven cell-layers at the positiveareaattheuninjectedsideandatthesidewhereGEF-H1
GEF-H1-MO-injectedside,incomparisonwithtwocell-layersin morpholino oligonucleotides had been injected (Fig. 4E,F; data
the uninjected tissue in the majority of the injected embryos not shown). These results suggest that GEF-H1 is required for
(n.30) (Fig. 4A–C; supplementary material Fig. S2A,B). Of early cell intercalations, which normally lead to dispersion of
note, the lateral plate mesoderm was also wider at the injected the clones before the midgastrula stage. Embryo cryosections
side.ThesameeffectswereseenwithGEF-H1aMO,butnotwith revealedfourtosixectodermalcell-layersingastrulatissuesthat
the control MO (data not shown). These observations suggest hadbeendepletedofGEF-H1;bycontrast,uninjectedtissueshad
that,inadditiontoapicalconstriction,GEF-H1isinvolvedincell onlytwotothreecell-layers(Fig. 4A–C;supplementarymaterial
intercalations,whichoccurbothinneuralandnon-neuraltissues. Fig. S2A,B). These changes were visible as early as stage 10 in
To exclude the possibility that GEF-H1 has an inhibitory effect allsectionedembryos(n535),indicatingthatGEF-H1isrequired
on cell divisions, we assessed the number of mitotic cells by in order for ectoderm to undergo radial intercalations. This
stainingembryosectionswithanantibodyagainstphosphorylated epiboly defect might, therefore, contribute to subsequent neural
histone 3 (H3), which recognizes only mitotic nuclei (Saka and tube closure.
Smith, 2001). The number of phosphorylated-H3-positive cells We next investigated whether GEF-H1 is involved in other
was not significantly different at the side that had been injected types of cell intercalatory movements, such as mesodermal
withthemorpholinooligonucleotideversusthatoftheuninjected convergent extension (Keller, 2002). To investigate whether this
side (supplementary material Fig. S2C,D). These results suggest cell behavior requires GEF-H1, we studied Xenopus ectodermal
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Fig.3. DepletionofGEF-H1interfereswithapicalconstrictionintheneuralplate.Four-cellembryoswereunilaterallyco-injectedwith40ngofmorpholino

oligonucleotideagainstGEF-H1(GEF-H1MO)and200pgofGFPRNA(asalineagetracer;green).Immunostainingoftransverseneuralfoldsections of
(instage-16embryos)forapicalconstrictionmarkers.Theuninjectedsideservedasacontrol(cost16).Arrowspointtotheuninjectedarea,*cellsthathadbeen al
injectedwithGEF-H1MO.Themidline(M)isindicatedbythebrokenline.(A,B)phosphorylatedmyosinIIlightchain(pMLC;red),(C,D)F-actin,(E,F)Rab11. n
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Control-MO-injectedembryos(supplementarymaterialFig.S1D–F)wereindistinguishablefromtheuninjectedembryos.B9,D9,F9showtheimagesinthe u
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correspondingpanelintheredchannelonly;B99andD99showtheimagesinthecorrespondingpanelinthegreenchannelonly.Scalebar:20mm. J
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Fig.4. GEF-H1functionstoregulateradialcellintercalations.(A)Embryonicectodermatstage16(st16)thathadbeeninjectedwiththecontrolmorpholino
oligonucleotide(co).(B)GEF-H1-depletedembryo(GEF-H1MO)containsabnormallythickectodermatstage16.GFPwasinjectedasdescribedintheFig.3
legend.(C)TheroleofGEF-H1inepiboly.Eight-cellembryos(n.20)wereinjectedwith20ngofGEF-H1MOwith150pgofmembraneassociatedGFPRNA
(mGFP;green)asatracerintoonedorsalanimal-poleblastomere.Cross-sectionofarepresentativeinjectedembryo(stage10)stainedforb-catenin
(red)tomarkcellboundaries.UptoeightcelllayersarevisibleatthesiteofGEF-H1MOinjection(arrows)ascomparedwiththeuninjectedside,whichcontains
3–4layers.Theanimalpoleisatthetop.Dashedlineindicatesmidline.DAPIstainingisblue.(D)SchemeofexperimentsshowninEandF.Four-cellembryos
wereinjectedunilaterallyinanimal-dorsalblastomerewiththeGEF-H1MO(40ng),followedbybilateralinjectionofLacZRNA(25pg)atthe16-cellstage.
Atstage11,clonedistributionwasrevealedbystainingforb-galactosidase(b-gal)activity.(E)ClonedispersionisdefectiveattheGEF-H1-depletedsideofthe
embryoatstage11(arrows).Tworepresentativeembryosareshown.Theanimalpoleistothetop,dorsalview.(F)Quantificationoftheexperimentshown
inDandE.Theresultsareshownasthemeandiametersoftheb-galactosidase-positiveareas6s.d.relativetothewholeembryosize,thenumberofembryos
thatwasinvestigatedisshownacrossthetop.StatisticalsignificancewasassessedbyusingStudent’st-test,***P,0.001.(G–J)GEF-H1isnotrequiredfor
animalcapelongationinresponsetoactivinA.Four-toeight-cellembryoswereinjectedintofouranimal-polesiteswith20ngofGEF-H1MOorthe
controlmorpholinooligonucleotide(CoMo)asindicated.Animalcapexplantswerepreparedatstage8andtreatedwithactivinA(0.5ng/ml).Explant
morphologywasvisualizedwhenuninjectedembryosreachedstage16.
explantelongationinresponsetoactivinA(amesoderminducing indistinguishable from the activity of Shroom, an actin-binding
factor), which is a useful test for morphogenetic cell behavior PDZ domain-containing protein that is known to trigger apical
(Howard and Smith, 1993; Sokol, 1996). Surprisingly, we constriction (Haigo et al., 2003; Hildebrand and Soriano, 1999)
observed no significant effect of GEF-H1 MO on activin-A- (Fig. 5C). The effects of Xenopus wild-type GEF-H1 and
induced explant elongation (Fig. 4G–J). By contrast, Xdd1 (a constitutively active forms of human and Xenopus GEF-H1
truncated form of Xenopus Dvl2) efficiently inhibited this weresimilar(datanotshown),suggestingthatwild-typeGEF-H1
behavior (Sokol, 1996; data not shown). These observations isnotinhibitedbymicrotubulesinourexperimentalsetting.The
support a selective role of GEF-H1 in radial intercalations and inactive forms of human and Xenopus GEF-H1, which have
apical constriction, but not in activin-dependent convergent mutations in the Dbl homology domain that abolish nucleotide
extension of mesodermal cells. exchangeactivityofGEF-H1(Krendeletal.,2002),didnotalter
the normal appearance of the injected ectoderm, demonstrating
GEF-H1triggersectopicapicalconstrictioninembryonic that the effect requires the Rho GEF activity of GEF-H1
ectoderm (supplementary material Fig. S3A–C; data not shown).
To initiate the analysis of the pathway used by GEF-H1, Examination of transverse cryosections confirmed that the
we wanted to establish a gain-of-function assay for GEF-H1 superficial cell layer that expressed GEF-H1 frequently
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activity in vivo. To prevent GEF-H1 inhibition by associated contained cells that were elongated along the apico-basal axis c
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microtubules,weusedconstitutivelyactivehumanGEF-H1with andcomprisedareducedapicalsurface(‘bottlecell’morphology) e
theC53Rmutation(GEF-H1-C53R)(Krendeletal.,2002).RNAs bycomparisontothatoftheuninjectedcontrolsorcellsthathad ci
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encoding GEF-H1-C53R or wild-type Xenopus GEF-H1 were beeninjectedwiththeinactiveformofhumanGEF-H1(Y393A)
microinjected into animal blastomeres of four-cell embryos. By (Fig. 5D–J; data not shown). These observations suggest that ell
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theonsetofgastrulation,bothRNAscausedtissuebendingatthe GEF-H1 is sufficient to stimulate ectopic apical constriction in
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site of injection, accompanied by increased pigment granule Xenopus embryonic ectoderm.
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accumulation (Fig. 5A,B). These morphological changes are To investigate how GEF-H1 triggers apical constriction, we n
characteristic of apical constriction (Lee and Harland, 2007; first determined its subcellular localization in ectodermal cells. ur
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Sawyer et al., 2010). Indeed, these effects of GEF-H1 were Astheendogenousproteinwasnotdetectedbyusingcommercial J
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Fig.5. GEF-H1inducesectopicapicalconstrictioninectodermalcells.(A–C)EctopicapicalconstrictioninducedbyGEF-H1andShroominectoderm.
Four-cellembryoswereinjectedwithRNAsandcultureduntillate-blastulaorearly-gastrulastages(9–10+).(A)Uninjectedcontrol(Co)embryo.(B)Embryo
injectedwithXenopusMyc–GEF-H1RNA(50pg).(C)EmbryoinjectedwithShroomRNA(200pg).Arrowspointtotheareasundergoingapicalconstriction.Top
viewisshown.(D–G)Cross-sectionsofembryosthathadbeeninjectedwithhumanGEF-H1-Y393A(D)orhumanGEF-H1-C53R(E)orShroom(F)RNA.
Ectodermcellsaccumulatepigmentattheapicalsurfaceandreducetheirapicalsurfaces(arrows).HumanGEF-H1-Y393A-expressingcellsare
indistinguishablefromcontroluninjectedectoderm.(G)Schemeoftheexperimentsshowingthetissuesectionedin(D–J),apicalisup.(H)Quantificationofcell
shapeinGEF-H1-expressingandcontrol(uninj)cells.Resultsareexpressedasratiosofapical-basalcelllengthoverapicalwidth(ABL/AW)inthesuperficial
layercells(asshowninDandE).Means6s.d.areshown.Thenumbersabovethebarsindicatethenumberofcellscountedpergroup(n).FiveGEF-H1-
expressingandtwocontrolembryoswereexamined.StatisticalsignificancewasassessedbyStudent’st-test,P,0.0001.(I)Morphologyofcellsthatunderwent
apicalconstriction(arrows)fromembryosthathadbeeninjectedwithXenopusMyc–GEF-H1(50pg)orShroom(200pg)RNAsasindicated.Thecell
membraneismarkedbyco-expressionofmembrane-targetedGFP(mGFP),whichwasdetectedbyusinganantibodyagainstGFP(green).Co,control
ectoderm.(J)EnfaceviewsofectodermfromtheexperimentinI.ArrowspointtocellsthatexpressXenopusMyc-GEF-H1orShroomwithmembrane-
associatedGFP(mGFP).Liveembryosareshown.*uninjectedcells.Scalebars:20mm.
antibodies, we visualized epitope-tagged GEF-H1 after it had resulted in the accumulation of c-tubulin, which has been
been expressed in embryonic ectoderm. Both Myc- and implicated in the formation of polarized microtubule arrays in
hemagglutinin (HA)-tagged GEF-H1 proteins were enriched cells that constrict in response to Shroom (Lee et al., 2007a)
near the apical surface of the constricting superficial cells, in (Fig. 6H,I).TheseresultssupportthepossibilitythatGEF-H1acts
addition to the cytoplasmic and cortical localization in both to locally activate actomyosin contractile filaments, which
superficial and inner ectoderm (Fig. 6A,B; data not shown). mediate apical constriction.
Inactive Xenopus Myc–GEF-H1 with the mutation Y444A
was mostly cytoplasmic with little cortical localization ROCKisinvolvedinGEF-H1-dependentapicalconstriction
(supplementary material Fig. S3D,E). These observations ROCK is a key effector of RhoA, which promotes the
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suggest that apically localized GEF-H1 promotes the formation phosphorylation of the regulatory MLC (Vicente-Manzanares c
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of the apical actomyosin contractile network that is essential for et al., 2009). We therefore tested the involvement of ROCK in e
apical constriction. We therefore stained for the presence of F- GEF-H1-induced apical constriction. An N-terminal truncation ci
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actin and phosphorylated MLC at the apical surface. Both form of ROCK, which acts in a dominant-negative manner
markers were detectable in hyper-pigmented cells that expressed (Marlowetal.,2002),efficientlyinhibitedtheapicalconstriction ell
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low amounts of GEF-H1, yet they were absent from the apical inducedbyeitherhumanorXenopusGEF-H1RNAs(Fig. 7A–D;
f
o
surface of the uninjected tissue (Fig. 6C,D,F,G).Consistent with data not shown). Alone, dominant-negative ROCK caused
these findings, the overexpression of GEF-H1 and ROCK also depigmentation of blastula ectodermal cells (Fig. 7C). The al
n
increased the levels of phosphorylated MLC in western blot levels of phosphorylated MLC were increased in ectodermal ur
assays (Fig. 6E). Additionally, the injection of GEF-H1 RNA cells that expressed active GEF-H1, but not in those that o
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Fig.6. GEF-H1activatesactomyosincontractilityin
superficialectoderm.(A,B)Cross-sectionsofsuperficial
ectodermthatexpressedXenopusMyc-GEF-H1RNA(25–50pg).
RNAinjectionswereasdescribedintheFig.5legend.
(A)Brightfieldimageofuninjectedectoderm(Co).(B)Brightfield
viewofGEF-H1-expressingcellsthatareelongatedandhighly
pigmented.(B9)Myc-stainingshowsapicalenrichmentofXenopus
Myc-GEF-H1inconstrictingcells,whichareelongatedandheavily
pigmented(arrow).(C,D)AccumulationofapicalF-actin(red)
revealedbyPhalloidinstaining(arrow)incross-sectionsof
XenopusMyc-GEF-H1-expressingectoderm(D),butnotin
uninjectedcontroltissue(C).(E)GEF-H1andROCKactivateMLC
phosphorylation(pMLC).Immunoblotanalysisoflysatesofstage-
11embryos,whichhadbeeninjectedwithGEF-H1andROCK
RNA,usingantibodiesagainstphosphorylatedMLC.Nochangein
totalGFP–MLClevelsisdetected(antibodyagainstGFP).
(F,G)AccumulationofpMLC(green)inGEF-H1-expressingcells.
(F)Controluninjectedembryonicectoderm.(G)Accumulationof
pMLCatconstrictedapicalsurfaces(arrows)ofXenopusMyc-
GEF-H1-expressingcells.G9,Brightfieldrevealselongated‘bottle
cell’morphology(arrows)withreducedapicalsurfacesand
increasedpigmentation.(H,I)GEF-H1inducesc-tubulin
redistribution(red).(H)Controlectodermatstage10.5showing
centrosomallocalization(arrowheads).(I,I9)Myc-GEF-H1RNA-
injectedectodermwithconstrictingcells(arrow).I9showsthered
channelonly.Scalebars:20mm.
expressed the inactive mutant (Fig. 6E–G and Fig. 7E). This constrictiondefects,theabnormalradialintercalationofGEF-H1
ability of GEF-H1 to stimulate phosphorylation of MLC was morphantsthatisrevealedatearly-gastrulastagesisprobablydue
also blocked by dominant-negative ROCK. Finally, we carried to the inhibition of MLC phosphorylation.
out transcriptional assays in Xenopus ectoderm cells that
transiently expressed the serum response factor (SRF) and DISCUSSION
luciferase (SRF-Luc) reporter (Krendel et al., 2002; Posern This study addressed the functions of GEF-H1, a RhoA-specific
et al., 2002; Posern and Treisman, 2006). GEF-H1 efficiently GEF, in Xenopus embryogenesis. The knockdown of GEF-H1
upregulated SRF-Luc but not the control reporter, which had resulted in neural tube defects, which are probably caused by
mutated SRF-binding sites. This reporter activation required abnormal cell intercalations and defective apical constriction in
ROCK (Fig. 7F). Taken together, these results strongly indicate GEF-H1-depletedneuroectoderm.Ingain-of-functionexperiments,
that GEF-H1 induces ectopic apical constriction through the we showed that GEF-H1 stimulates ectopic apical constriction in
activation of the RhoA–ROCK–myosin-II signaling pathway. Xenopusectoderm.Takentogether,ourexperimentsdemonstratea
crucial role for GEF-H1 in cell behaviors that lead to vertebrate
GEF-H1controlsintercalatorybehaviorbymodulating neuraltubeclosure.Theseobservationsdifferfromthosethathave
myosinIIactivityindeepectodermalcells been previously reported for GEF-H1. One study has reported a
To further investigate how GEF-H1 regulates cell intercalations, modesteffectofmurineGEF-H1/Lfconectodermalcompetencein
we assessed the staining of phosphorylated MLC in deep responsetoneuralinducers(Morganetal.,1999).Twootherstudies
ectoderm cells, in which we have manipulated GEF-H1 activity have proposed a role for GEF-H1/Lfc in inhibiting convergent
ingain-andloss-of-functionexperiments.Atstages10–11,deep extensionmovementsinresponsetomicrotubuledepolymerization;
ectoderm cells exhibitedpolarized MLC phosphorylation,witha however, the depletion of the reported GEF-H1 variant did not
lack of staining in the basal domain (Fig. 8A; supplementary perturb normal embryonic development (Kwan and Kirschner,
materialFig.S4).GiventhekeyroleofphosphorylatedMLCand 2005;Zhouetal.,2010).BecausethepreviouslydescribedGEF-H1
myosin II in cell intercalations, this polarity might reflect the cDNA sequence is not present in the National Center for
e
ability of these cells to intercalate between cells that are located Biotechnology Information (NCBI) databases, it is likely to c
n
more apically. We observed that overexpressed GEF-H1 marked encodearareisoformoftheprotein. e
apical cell protrusions that did not contain phosphorylated MLC Apical constriction is a morphogenetic process that underlies ci
S
(Fig. 8B).Suchprotrusionsweremuchmorefrequentincellsthat epithelial tissue folding in diverse developmental contexts,
expressed GEF-H1 by comparison with those that had not been including neural tube closure (Lee and Harland, 2007; Sawyer ell
C
injected(morethan30embryosexamined),indicatingthatGEF- et al., 2010). GEF-H1-initiated apical constriction is ROCK-
f
H1 stimulates the protrusive activity in deep ectodermal cells. dependent and leads to myosin II activation. So far, only a o

Moreover,inGEF-H1-MO-injectedcells,butnotincontrol-MO- handful of proteins have been shown to trigger ectopic apical al
n
injected cells, phosphorylated MLC staining was disorganized constriction.OneoftheseproteinsisShroom(Haigoetal.,2003; ur
(Fig. 8C,D). These observations indicate that, similar to apical Hildebrand, 2005; Hildebrand and Soriano, 1999), which has o
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Fig.7. ROCKisrequiredforGEF-H1-inducedectopicapicalconstriction.(A,B)Four-cellembryoswereinjectedwithGEF-H1-C53R(60pg)and/or
dominant-negativeROCK(DN-ROCK,400pg)RNAsintotwoanimal-ventralblastomeres.Theanimalpoleviewofstage-8.5embryosisshown.(A)Intense
pigmentation(arrows)accompaniesapicalconstrictioninresponsetoexpressionofGEF-H1-C53R.(B)Dominant-negativeROCKcompletelyblockstheeffect
ofactiveGEF-H1.(C)QuantificationofphenotypicchangesforembryosthathadbeeninjectedwiththeindicatedRNAs.Scoring:++,hyper-pigmentation;
+,weakhyperpigmentation;nochange,normalmorphology;2,de-pigmentation.Pigmentaccumulationwasassociatedwithapicalsurfacereductionand
constriction.They-axisshowsthepercentageofembryoswiththeindicatedphenotype.Thenumbersacrossthetopindicatethetotalnumberofembryos
examined.(D)ProteinlevelsforGFP-GEF-H1-C53RandMyc-taggeddominant-negativeROCKwereassessedbyusingantibodiesagainstGFPandMycin
embryolysatestakenfromexperimentsperformedinAandB.(E)Dominant-negativeROCKsuppressesGEF-H1-dependentMLCphosphorylation(pMLC).
Four-cellembryoswereinjectedintofouranimalblastomereswithRNAsencodingGFP–MLC(250pg),GEF-H1-C53R(C53R,60pg),GEF-H1-Y393A
(Y393A,500pg),dominant-negativeROCK(400pg,H,highdose;200pg,L,lowdose),orwild-typeROCK(ROCK,250pg)asindicated.Injectedembryos
werelysedatstage8.5forwesternblottinganalysiswithantibodiesagainstpMLC.AntibodystainingagainstMycandGFPshowsthelevelsofdominant-
negativeROCKandGFP–MLC,respectively.a-tubulinisloadingcontrol.(F)ROCKisrequiredforGEF-H1-mediatedSRF-Lucreporteractivation.20pgofSRF
reporter(3DA-SRF-Lucormutated2MA-SRF-Luc)DNAswereco-injectedwithRNAsencodingGEF-H1-C53R(60pg)anddominant-negativeROCK(250pg)
intotwodorsal-animalblastomeresoffour-cellembryos,asindicated.Therelativeluciferaseactivitywasmeasuredinlysatesofembryosharvestedat
stage10.5.Means6s.d.areshown.Everygroupincludedfourindependentsets,eachcontainingfourembryos.
been proposed to induce apical constriction through recruiting and stimulate p114GEF (also known as ARHGEF18 in human),
ROCK (Mohan et al., 2012; Nishimura and Takeichi, 2008). leading to ROCK and myosin II activation (Nakajima and
e
During chicken lens pit invagination, the ability of Shroom to Tanoue, 2012). It will be important to confirm the connection c
n
drive apical constriction has been linked to RhoA and the RhoA between Lulu and p114GEF in diverse experimental models and e
GEF Trio (Plageman et al., 2011). Our analysis revealed to test the alternative possibility that Lulu regulates neural tube ci
S
remarkable similarities between GEF-H1 and Shroom, both in closurethroughGEF-H1.Ofnote,wehavepreviouslyfoundthat
gain- and loss-of-function experiments, suggesting that the two Xenopus Lgl1, a polarity protein that binds myosin II, also ell
C
proteinsfunctioninthesamemolecularpathway.Anotherprotein induces apical constriction and that this activity is under the
f
o
thatisinvolvedinbothapicalconstrictionandneuraltubeclosure control of Wnt signaling, but the mechanism of this regulation,
is Lulu, also known as the FERM-domain-containing protein and its possible connection to GEF-H1, remains to be clarified al
n
Epb4.1l5 (Chu et al., 2013; Lee et al., 2007b; Nakajima and (Choi and Sokol, 2009; Dollar et al., 2005). Interestingly, GEF- ur
Tanoue, 2012). In cultured mammalian cells, Lulu can bind to H1 has been implicated in Wnt3a-dependent neurite retraction o
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RESEARCHARTICLE JournalofCellScience(2014)127,2542–2553doi:10.1242/jcs.146811
Fig.8. GEF-H1regulatesprotrusivebehaviorandthe
polarizeddistributionofphosphorylatedmyosinlight
chainininnerectodermcells.(A–D)Immunostainingof
transverseectodermsectionsofstage-11(st11)embryosthat
hadbeeninjectedwiththeindicatedmorpholino
oligonucleotides(40ng)orMyc–GEF-H1mRNA(5–10pg).
GFPisalineagetracer.(A)Incontrol(Co)ectoderm,
phosphorylatedMLC(pMLC)isenrichedattheapico-lateral
boundariesofinnercells(arrowhead).Theapical–basalaxis
isindicated.(B)OverexpressionofMyc–GEF-H1leadstothe
formationofapicalGEF-H1-positiveprotrusionsininner
ectodermcells(arrow,n.25).pMLCstainingisshown(red).
B9showsthestainingofMyc(green)inthesamefieldasthat
showninB.B0showsthemergedimageofBandB9.
(C,D)TheGEF-H1morpholinooligonucleotide(GEF-H1MO,
D)butnotthecontrolmorpholinooligonucleotide(CoMO,C)
interfereswithpMLC(red)polarizationandcauses
disorganizedinnercellmorphologyintheectodermof
injectedembryos(n.25).C9andD9showmergedimagesof
thepMLCstainingfromthecorrespondingpanelwith
localizationofGFP.*lackofpMLCstaininginGEF-H1-
depletedcellsascomparedwiththatofuninjectedcells
(arrow).Scalebar:20mm.
and found to interact with the components of the Wnt pathway, Our results establish roles for GEF-H1 in multiple signaling
mammalian DVL1 and DAAM1 (Tsuji et al., 2010). processes that regulate cell shape and cell movements during
Besides apical constriction, ectoderm cell intercalation is also neural tube closure. Surprisingly, mice lacking the GEF-H1/Lfc
altered in GEF-H1 morphants at the gastrula and neurula stages. gene do not exhibit neural tube abnormalities (Meiri et al.,
MultiplecelllayerswereobservedinGEF-H1-depletedembryos, 2012), suggesting that this protein functions redundantly with
even before the onset of gastrulation, supporting the hypothesis other GEFs in neural tube closure. The pleiotropic roles of
that GEF-H1 is necessary for epiboly, which is largely driven GEF-H1 in cell intercalations and apical constriction parallel
by the radial intercalation of deep cells (Keller, 1980). These the known functions of myosin II, reiterating the view
early epiboly defects might be later responsible for abnormal that myosin II is a major cellular target of GEF-H1. Indeed,
neural tube closure in GEF-H1-depleted embryos. Alternatively, the changes in phosphorylated MLC that we observed upon
these neural tube defects might directly result from inhibited modulation of GEF-H1 indicate that GEF-H1 exerts its effect
dorsal convergence at late-gastrula and/or early-neurula stages. on both cell intercalation and apical constriction by regulating
Surprisingly, we found that ventral injections of GEF-H1 MO myosin II activity. Nevertheless, the involvement of GEF-H1 in
alsoledtoincompleteneuraltubeclosure.Thissuggeststhatnon- diverse cell behaviors might, in principle, be due to alternative
neural ectoderm, which fails to converge towards the midline in molecular targets (Birkenfeld et al., 2008). Besides its direct
GEF-H1-depleted embryos, might be partly responsible for the regulation of RhoA, GEF-H1 binds to Cingulin to regulate
neuralplatedefects(datanotshown).Attestingtothespecificity tight junctions (Aijaz et al., 2005; Benais-Pont et al.,
of GEF-H1 in regulating ectodermal cell behaviors, mesodermal 2003; Guillemot et al., 2008). Moreover, GEF-H1 has been
convergent extension of activin-induced animal pole explants identified as a molecular substrate for PAR-1, a component
e
was not inhibited by GEF-H1 MO. One potential explanation of the apical-basal polarity network (Yamahashi et al., c
n
of this result is that GEF-H1 is required specifically for 2011; Yoshimura and Miki, 2011). Based on the interaction e
neural convergent extension, whereas WGEF, an unrelated Rho of GEF-H1 with the exocyst component Sec5, Pathak and ci
S
GEF, functions to regulate mesodermal convergent extension colleagues (Pathak et al., 2012) proposed that the local
(Tanegashima et al., 2008). Although mediolateral cell activation of RhoA by GEF-H1 stimulates exocytosis. ell
C
intercalations are thought to drive dorsal convergence both Consistent with these findings, we observed that the apical
f
o
in mesoderm and neuroectoderm, the mechanisms that regulate accumulation of Rab11 in the neural plate is lost in GEF-H1-
this process in different germ layers are poorly understood (Elul depleted cells. Whereas Rab11 is a strong marker of apical al
n
et al., 1997; Keller et al., 1992a; Keller et al., 1992b; Keller, constriction (Ossipova et al., 2014), the causal relationship ur
1980). between GEF-H1-mediated Rab11 recruitment and RhoA o
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activation is, presently, unclear. Future studies are needed comparingtheaveragediametersofb-galactosidase-positiveareasonthe
to determine which of the identified molecular targets are left(control)andontheright(injectedwithmorpholinooligonucleotide)
involved in the regulation of specific morphogenetic cell sides,relativetothewholeembryolength.
EctopicapicalconstrictionwasexaminedatblastulastagesafterGEF-
behaviors by GEF-H1.
H1 RNA injections into two animal ventral blastomeres of 4–8 cell
embryos.Eachgroupcontained15to30embryosinseveralindependent
MATERIALSANDMETHODS experiments.OptimaldosesofRNAsweredeterminedinseparatedose-
DNAconstructsandmorpholinos response experiments for individual RNAs. Whole-mount in situ
The clone for Xenopus laevis GEF-H1 was obtained from Open hybridization with an antisense Sox2 probe (Mizuseki et al., 1998) was
Biosystems (GenBank accession number BC072246). Plasmids performedwhentheinjectedembryosreachedstages14–15,asdescribed
encoding the human GEF-H1-C53R and human GEF-H1-Y393A, previously (Harland, 1991). Neural tube defects were quantified by
which were fused to enhanced GFP (Krendel et al., 2002) were a gift comparingthewidthoftheSox2expressiondomainatthemid-hindbrain
fromCelineDermardirossian(ScrippsResearchInstitute,LaJolla,CA). level for the injected and the uninjected sides. Images were taken by
These constructs were subcloned into pXT7 for RNA injections. Pfu-I- usingaLeicastereomicroscopeandusingtheOpenlabsoftware.
directed mutagenesis, followed by DpnI digestion, was performed to For animal cap assays, injections were carried out into four animal-
generate wild-type human GEF-H1 and Xenopus Myc–GEF-H1-Y444A pole blastomeres at the 4–8-cell stage. When control embryos reached
asdescribedpreviously(Makarovaetal.,2000).XenopusGEF-H1a-HA, stage 8, ectodermal explants were excised and cultured in 0.16MMR
with and without morpholino oligonucleotide target sequences, were containing 0.1mg/ml bovine serum albumin with or without 0.5ng/ml
constructed by using 59-GGTGAATCGGGGATCGTACCCATACG- activin A. The explants were cultured until stages 15–16 to observe
ATGTTCCAGATTACGCTTCGGAGAGTTAAAGATGC-39and59-CG- elongation phenotypes. Each group consisted of 10–15 explants. These
CGTCCGGGCGTATACTCAATGGTCGGTGGGAAATCC-39 primers, experimentswererepeatedthreetimes.
respectively.Myc-GEF-H1wasconstructedbysubcloningthefull-length
XenopusGEF-H1ainsertintopCS2-Myc.TogenerateGFP-MLC-pXT7,an Immunostainingandcellshapeanalysis
insertcorrespondingtochickenMLCfusedwithGFP(Wardetal.,2002) For cryosectioning, embryos were devitellinized at the necessary stage,
wassubclonedintothevectorpXT7.Theotherplasmidsconstructswere fixedinMEMFAorDent’sfixativefor1–2hours,andembeddedin15%
pCS2-LacZ, the plasmid pCS2 encoding nuclear LacZ (Turner and fishgelatinand15%sucrosesolutionasdescribedpreviously(Ossipova
Weintraub, 1994); pCS2-memRFP, encoding membrane associated RFP etal.,2007).Theembeddedembryoswerefrozenindryiceandsectioned
(agiftfromSeanMegason,HarvardMedicalSchool,Boston,MA);pCS2- at 10–12mm using a Leica CM3050 cryostat. The cell shape in
mGFP, encoding membrane associated GFP (Ossipova et al., 2007); neuroectodermwasanalyzedincryosectionsthathadbeenstainedwith
wild-typeanddominant-negativeROCK(Marlowetal.,2002);andpCS2- anantibodyagainstb-catenin,whichmarkscellboundaryofallcellsat
ShroomL,encodingthelongformofShroom3(Hildebrandand Soriano, neurulastages.Theshapeofneuralplatecellswasscoredastheratioof
1999).Detailsofcloningareavailableonrequest. theapical-basallengthtotheapicalwidthfor5to7superficialcellsthat
NCBIdatabasesearchesidentifiedtwoXenopuslaevisGEF-H1cDNAs wereadjacenttothedorsalmidline.
(accessionnumbers:BC072246andNM_001136164,whicharereferredto Forindirectimmunofluorescence,embryosectionswerestainedwitha
asGEF-H1aandGEF-H1b,respectively,inthisstudy).Thetwoformsof rabbitantibodyagainstb-catenin(SantaCruz,1:400),arabbitantibody
GEF-H1 diverge only in the 59-untranslated sequence and several N- against phosphorylated histone 3 (Cell Signaling, 1:200), a mouse
terminal amino acids, probably due to alternative splicing. RT-PCR antibody against Myc (clone 9E10) (Roche, 1:50 from hybridoma
analysis revealed expression of GEF-H1a RNA in gastrula ectoderm as supernatant), a mouse antibody against GFP (clone B2, Santa-Cruz,
previouslyreported(Morganetal.,1999),butGEF-H1bRNAwaspoorly 1:200), a rabbit antibody against c-tubulin (Abcam, 1:100), a rabbit
detectable under the same conditions (data not shown). Therefore, antibody against Rab11 (Invitrogen, 1:100), a rabbit antibody against
subsequent analysis was restricted to GEF-H1a. For knockdown studies, HA (Bethyl Laboratories, 1:2000) and Alexa-Fluor-488-, Alexa-Fluor-
threedifferentmorpholinooligonucleotideswereobtainedfromGeneTools 555-conjugated (Invitrogen, 1:200) or Cy3-conjugated (Jackson
(Ekker,2000;Heasmanetal.,2000)withthefollowingsequences:GEF- Immunoresearch, 1:200) secondary antibodies. F-actin was detected by
H1aMO,59-AGGTGCGGTCAATGCCGGACATTGA-39;GEF-H1MO, using Alexa-Fluor-568-conjugated phalloidin (4 units/ml, Invitrogen).
59-ACATTGAGACGGATTTGGACAGAGG-39; GEF-H1b MO, 59- RFP fluorescence was visualized directly without immunostaining.
GGACCACCCTGTTGCTGCATCTTCT-39. These differ from GEF-H1/ Sections were stained with 1mg/ml DAPI and mounted by using
Lfc morpholino oligonucleotide sequences that have been reported Vectashieldmountingmedia(Vector).ForphosphorylatedMLCstaining,
previously(KwanandKirschner,2005;Zhouetal.,2010).Thesequence byusingarabbitantibodyagainstphosphorylatedMLC(Abcam,1:100),
ofthecontrolMOwas59-GCTTCAGCTAGTGACACATGCAT-39. gastrula-stageembryoswerefixedin2%trichloroaceticacid(TCA)for
30minandwashed inPBS+0.3%TritonX-100for30min(Nandadasa
Embryoculture,microinjections,clonalanalysis,insitu etal.,2009).ForMyc-orHA-taggedGEF-H1localization,embryoswere
hybridizationandanimalcapassay fixed, cryosectioned and subjected to indirect immunofluorescence.
Eggs and embryos were obtained from Xenopus laevis and cultured in Similarproteinlocalizationpatternswereobservedfordifferentlytagged
0.16Marc’smodifiedRinger’ssolution(MMR)(NewportandKirschner, GEF-H1 constructs using either Dent’s or TCA fixatives (data not
1982)asdescribedpreviously(Itohetal.,2005).Stagingwasperformedas shown).Stainingpatternswerereproducedinatleastthreeindependent
described previously (Nieuwkoop and Faber, 1967). For microinjection, experiments. Representative sections of experimental groups (each
embryosweretransferredto3%Ficollin0.56MMRandinjectedatthe4– containing 10–15 embryos) are shown. Imaging was performed by
8-cellstagewith5–10nlofasolutioncontainingRNAsand/ormorpholino usingaZeissAxioImagermicroscope. e
c
oligonucleotides. Capped synthetic RNAs were generated by in vitro n
e
transcription with SP6 or T7 RNA polymerase using the mMessage Westernblottinganalysisandluciferaseassays ci
mMachinekit(Ambion)fromthecorrespondingDNAtemplates. Western blotting analysis was performed using standard techniques as S

Forclonalanalysis,morpholinooligonucleotidesagainstGEF-H1were describedpreviously(Itohetal.,2000).Four-celltoeight-cellembryos ell
injected into one dorsal blastomere of four-cell embryos, followed by were injected with RNAs encoding GFP–GEF-H1-C53R, GFP–GEF- C
injections of LacZ RNA into two dorso-lateral blastomeres of 16-cell H1-Y393A, GFP–MLC, wild-type or Myc–dominant-negative-ROCK. f
o
embryos. b-galactosidase staining was carried out with Red-Gal Wholelysateswerepreparedfromfiveembryosatstage8.5or10.5–11.
al
(Research Organics) and reinforced by bleaching in 1% H2O2, 5% For testing morpholino oligonucleotide specificity, four animal-pole n
formamide in 0.56 saline sodium citrate buffer, following standard blastomeresof4–8-cellembryoswereinjectedwithXenopusGEF-H1- ur
protocols(Harland,1991).Clonespreadingwasquantifiedatstage11by HA RNA6GEF-H1a MO, GEF-H1 MO or control MO, and whole o
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