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INTRODUCTIONThe Xenopus blastula is a hollow ball of cells (blastomeres) with noskeletal tissue and little extracellular matrix. At this early stage,shape and rigidity of the whole embryo are provided by the corticalactin skeleton of each component blastomere. In the absence ofcortical actin, the embryo collapses under its own weight and themovements of gastrulation do not occur (Kofron et al., 2002; Tao etal., 2005). The mechanism by which the cortical actin skeleton isgenerated and maintained is therefore an important problem in earlydevelopment.

We have shown previously that the pattern and density of corticalactin changes through the cell cycle. During interphase, eachblastomere possesses a dense cortical actin network, which isreplaced during mitosis by a much sparser network of actin filamentbundles (Lloyd et al., 2005). Dissociation of the blastomeres, so thatcell contact and intercellular signals are prevented, causes allblastomeres to adopt the sparse configuration of actin, which isreversible by reassociation (Lloyd et al., 2005). Previous expressionscreening experiments identified two proteins that are required forassembly of cortical actin. Receptors for the signaling phospholipidlysophosphatidic acid (LPA) and an orphan G-protein-coupledreceptor (GPCR) named Xflop, were each found, by gain- and loss-of-function experiments, to be both necessary and sufficient forassembly of the dense cortical actin network (Lloyd et al., 2005; Taoet al., 2005). These experiments indicated the novel fact thatintercellular signaling plays a major role in generating theappropriate density and pattern of cortical actin assembly in the earlyembryo.

It therefore becomes important to determine the sites of actinassembly at the cell surface and the way the G-protein-coupledreceptors control the amount and pattern of actin assembly, so as togenerate the appropriate shape and mechanical rigidity of the wholeembryo.

Recent elegant experiments using epithelial cells in culture haveshown that during initial contacts between epithelial cells thecytoplasmic domains of transmembrane cadherins are sites of actinassembly (Ehrlich et al., 2002; Jamora and Fuchs, 2002; Kovacs etal., 2002a; Kovacs et al., 2002b; Vaezi et al., 2002). E-cadherinengagement in cultured cells causes activation of Rac1 and Cdc42,both of which are involved in actin polymerization and organization(Betson et al., 2002; Kim et al., 2000; Kovacs et al., 2002a;Nakagawa et al., 2001; Noren et al., 2001). Actin nucleation proteinssuch as members of the Arp2/3 complex and formin 1 are associatedwith nascent cadherin-mediated adhesive contacts (Kobielak et al.,2004; Kovacs et al., 2002b; Verma et al., 2004), as are nucleation-promoting factors such as Ena/Vasp and cortactin (Helwani et al.,2004; Scott et al., 2006; Vasioukhin et al., 2000). However, thedetailed mechanism by which cortical actin assembles at thecadherin complex is still unknown (Adams and Nelson, 1998; Dreeset al., 2005; Perez-Moreno et al., 2003; Yamada et al., 2005).

Could cadherins be the sites of assembly of cortical actin in theXenopus blastula? It is known that the early blastomeres are heldtogether by calcium-dependent adhesion (Nomura et al., 1988;Turner et al., 1992). The calcium-dependent adhesion protein C-cadherin (also known as EP-cadherin) (Choi et al., 1990; Ginsberget al., 1991) is expressed during the egg-to-blastula stages and hasbeen shown, by antisense-mediated mRNA depletion, to beabsolutely required for cell adhesion at this stage (Heasman et al.,1994).

In this paper, we show that C-cadherin is essential for theassembly of the dense cortical network seen in interphaseblastomeres, but not for the sparse network seen during celldivisions. First, we show that C-cadherin proteins are organized aspunctae on the surfaces of the blastomeres and that the actin filamentnetwork is associated with these punctae. Second, we show by bothgain- and loss-of function experiments that the level of C-cadherin

G-protein-coupled signals control cortical actin assembly bycontrolling cadherin expression in the early Xenopus embryoQinghua Tao1,*, Sumeda Nandadasa1,2,*, Pierre D. McCrea3, Janet Heasman1 and Christopher Wylie1,†

During embryonic development, each cell of a multicellular organ rudiment polymerizes its cytoskeletal elements in an amount andpattern that gives the whole cellular population its characteristic shape and mechanical properties. How does each cell know howto do this? We have used the Xenopus blastula as a model system to study this problem. Previous work has shown that the corticalactin network is required to maintain shape and rigidity of the whole embryo, and its assembly is coordinated throughout theembryo by signaling through G-protein-coupled receptors. In this paper, we show that the cortical actin network colocalizes withfoci of cadherin expressed on the cell surface. We then show that cell-surface cadherin expression is both necessary and sufficientfor cortical actin assembly and requires the associated catenin p120 for this function. Finally, we show that the previously identifiedG-protein-coupled receptors control cortical actin assembly by controlling the amount of cadherin expressed on the cell surface.This identifies a novel mechanism for control of cortical actin assembly during development that might be shared by manymulticellular arrays.

KEY WORDS: Cortical actin, Actin assembly, Xenopus, GPCR, Cadherins, C-cadherin, p120 catenin, Xflop, LPA

Development 134, 2651-2661 (2007) doi:10.1242/dev.002824

1Children’s Hospital Research Foundation, Division of Developmental Biology,Cincinnati, OH 45229, USA. 2Molecular and Developmental Biology GraduateProgram, University of Cincinnati College of Medicine, OH 45219, USA.3Department of Biochemistry and Molecular Biology, Program in Genes andDevelopment, University of Texas Graduate School of Biomedical Sciences, Houston,TX 77030, USA.

*These authors contributed equally to this work†Author for correspondence (e-mail: Christopher.Wylie@cchmc.org)

Accepted 8 May 2007

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expression is both necessary and sufficient for the dense corticalactin network. Increasing the level of C-cadherin expression leadsto increased cortical actin density, whereas decreasing C-cadherinexpression, by depletion of its mRNA, reduces it. We also show thatthe juxtamembrane domain of C-cadherin, which binds p120catenin, is essential for its control of actin assembly. Consistent withthis, we show that p120 catenin is required for dense actin networkassembly and that a mutant form of p120 catenin that lacks thebinding site for C-cadherin fails to increase actin assembly. Thesedata show that the cadherin complex is both necessary and sufficientfor assembly of the normal pattern and density of cortical actin inthe Xenopus blastula, and that this activity requires p120.

If cortical actin assembly depends on the expression level of C-cadherin, then LPA signaling, and signaling through the G-protein-coupled receptor Xflop, could act either at the level of actinnucleation, or at the level of C-cadherin presentation on the cellmembrane (or both). We show here that both signaling pathwayscontrol the level of C-cadherin expression on the cell surface. Thesedata show for the first time how C-cadherin levels on the cell surfaceare controlled in the Xenopus embryo and the consequences of thiscontrol for actin assembly.

MATERIALS AND METHODSOocytes and embryosAll frogs (Xenopus laevis) were obtained from Nasco (Fort Atkinson, WI).Full-grown oocytes were obtained by manual defolliculation and cultured inoocyte culture medium (OCM). To prepare 800 ml OCM: 480 ml LeibpvitzL-15 with glutamine (Sigma Aldrich), 320 ml water, 4 ml Pen-Strep Solution(MP Biochemicals, LLC), 320 mg BSA and adjust pH to 7.6-7.8 using 5 MNaOH. Antisense oligonucleotides or synthetic mRNAs (doses as indicatedin text) were injected into defolliculated oocytes. After oligonucleotideinjection, oocytes were cultured for 2-3 days before maturation. AftermRNA injection, oocytes were matured overnight, starting on the day of theinjections. Progesterone (1-2 �M) was used to mature oocytes. Maturedoocytes were fertilized by the host transfer technique (Heasman et al., 1991).

DNA constructs and RNATo generate a p120 catenin mRNA to which the MO could not bind, wemade a cDNA construct with three third-base alterations using the followingprimer pair for PCR cloning: forward, 5�-CGGAATTCTATG GA -CGAACCTGAGTCTGAAAGTCCG-3� (underlined are the EcoRI site forsubcloning, the ATG start codon, and the three substitutions at the third baseof each codon, respectively); reverse, 5�-TACGCAGGCA ACCTGTAGTG-3�. Xflop R112A mutant was generated by PCR-based mutagenesis, usingthe following PCR primers (underlined nucleotides are designed to substitutearginine with alanine):Xflop5�-BglII, 5�-AGCAGATCTATGGCGTGTAATCAGAGCTGTGA -ATAC-3�;R112A r, 5�-CACTGTAGCCACAAAAGCATCCATGGCTATACAGCT-3�;R112A f, 5�-TGTATAGCCATGGATGCTTTTGTGGCTACAGTGTTC-3�;andXflop3�-EcoRI, 5�-GTTGAATTCTATCCTGTCCTTTTTGATGAC CTC -CTTC-3�.

The primer pair of Xflop5�BglII and R112A r was used to generatefragment 1 using wild-type Xflop cDNA as template, whereas the primerpair of R112A f and Xflop3�EcoRI was used to generate fragment 2 usingwild-type Xflop cDNA as template. The primer pair of Xflop5�BglII andXflop3�-EcoRI was then used to generate the full-length Xflop R112Amutant using the purified fragments 1 and 2 as template. The full-lengthXflop R112A was inserted into BamHI and EcoRI-cut pCS107 vector. AllPCR products were verified by sequencing.

A Xenopus tropicalis C-cadherin construct was isolated from an arrayedcDNA library constructed in vector pCS107 (a gift from Aaron Zorn,University of Cincinnati, OH), linearized with Asp718, then transcribed withSP6 RNA polymerase. Xenopus C-cadherin mutants [C-cad(G-A)-HA

(referred to below as C-cad G-A), C-cad �CBD] (Paulson et al., 2000) weresynthesized with SP6 RNA polymerase from a pCS2+ vector linearized withNotI. Xenopus p120 iso1, p120 iso1 MO-resistant and the Arm1 deletionmutant (p120 �Arm1) were constructed in pCS2-MT vector with a Myc tagat the N-terminus of p120. For RNA synthesis, we linearized the vector withNotI and transcribed from the SP6 promoter. pCS107 Xflop and XflopR112A and LPA2 were each linearized with Asp718 and transcribed withSP6 RNA polymerase. Message Machine (Ambion) Kits were used for allin vitro transcriptions.

OligonucleotidesThe antisense oligodeoxynucleotides (designated AS below) or morpholinooligonucleotides (designated MO below) used were (asterisks indicatephosphorothioate-modified residues):C-cad AS61 (Heasman et al., 1994), 5�-C*C*T*CTCCAGCTC -CCT*A*C*G-3�;P120 catenin AS1, 5�-TGCATCCCTCCATCCTGT-3� (no modification);P120 catenin MO1 (Fang et al., 2004), 5�-ACTCTGGCTCATC -CATATAGAAAGG-3�;Xflop oligonucleotides (Tao et al., 2005) 1s, 5�-A*A*G*GGA -ACACTGTAG*C*C*A-3� and 5s, 5�-G*T*T*GTACGTTTTGGC*T* -G*G-3�;LPA1 MO (Lloyd et al., 2005), 5�-TTCACTTCAGATGTC -AGTCATGCTG-3�; andLPA2 MO (Lloyd et al., 2005), 5�-ACCTCCAATGTTACAGC -GCAGCCTC-3�.

Immunofluorescence and F-actin stainingFor F-actin single staining, caps were excised at the late blastula stage (St9)(Nieuwkoop and Faber 1967) and fixed with 3.7% formaldehyde, 0.25%glutaraldehyde in PBS, 0.1% Tween 20 (FG fix) for 10 minutes. Caps werethen washed three times for 10 minutes each and stained with 5 U/ml Alexa488-conjugated phalloidin (Molecular Probes, Oregon) for 3 hours at roomtemperature or overnight at 4°C. For anti-C-cadherin/F-actin doublestaining, the phalloidin-stained caps were blocked with 10% normal goatserum at room temperature for 1 hour and then incubated with 5 ng/ml anti-C-cadherin monoclonal antibody (6B6, Developmental Study HybridomaBank, Iowa) overnight at 4°C, followed by extensive washing with PBS,0.3% Triton X-100. A Cy5-conjugated goat anti-mouse IgG (JacksonLaboratory, 1:300) was then added, followed by 2 hours incubation at roomtemperature. In every experiment, 3-5 caps were also incubated withsecondary antibody only as negative controls. For anti-C-cadherin singlestaining, 2% TCA in water was used to fix animal caps for 30 minutes atroom temperature. After anti-C-cadherin staining, caps were dehydrated ina methanol series and cleared in Murray’s Clear [from Murray andKirschner, as cited by Dent and Klymkowsky (Dent and Klymkowsky,1989)], before examination by confocal microscopy.

Confocal microscopy and data acquisitionA Zeiss LSM 510 confocal microscope was used. A Fluar UV/20�NA0.75objective was used for lower magnification imaging, and a C-Apochromat63�/NA1.3 water lens or a Plan-Neofluar40�/NA1.3 oil lens was used with1.5-2� digital zoom for higher magnification imaging. All images weregained with the size of 512�512 pixels (8 bit for experiments representedin Figs 6-8, 12 bit in experiments represented in Figs 2-5, 9). The intensityof phalloidin staining of each pixel measured by LSM510 software was usedto quantify the levels of F-actin. The level of F-actin for each imaged areawas the average (Mi) of intensity measurements from all 512�512 pixels.The level of F-actin for each experimental treatment group was the averageof 5-10 animal caps (Mt=�Mi/i, where i=number of imaged areas). Thelinear range of pixel intensity measurement was from 0 to 255 for 8-bitimages or from 0 to 4095 for 12-bit images. In all experiments, the confocalsettings were optimized to allow more than 90% of pixels to have intensitymeasurement within the linear range, which might introduce variationsamong control groups from experiment to experiment. Each histogramshown in Figs 2-9 represents the mean pixel intensity (Mt) ±s.d. from 5-10animal caps. The two-tailed t-test was used to generate P values.

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Western blottingTo detect the total protein levels of p120 or C-cadherin, five embryos werehomogenized with 250 �l of ice-cold PBS, 1% Triton X-100 containing 1 mMPMSF and a 1:100 dilution of protease inhibitor cocktail (PIC, Sigma P8340),and cleared by centrifugation at 750 g for 10 minutes at 4°C. The supernatantswere then transferred into precooled Eppendorf tubes. An equal volume of 4�sample buffer was added to the cleared supernatants.

To detect the membrane levels of C-cadherin or its binding proteins,including p120, plakoglobin and �-catenin, five embryo samples werehomogenized in volumes of 50 �l per embryo in ice-cold membranepreparation buffer (250 mM sucrose, 10 mM HEPES-NaOH, 2 mM MgCl2, 1mM EGTA, 0.5 mM EDTA, 1 mM PMSF, 1:100 dilution of PIC). Lysateswere cleared by centrifugation at 700 g for 10 minutes at 4°C. Supernatantswere transferred into precooled clean tubes and centrifuged at 21,000 g for 30minutes at 4°C. Pellets were resuspended with 30 �l of 2� sample buffer.

Two to three embryo equivalents of membrane protein, or one embryoequivalent of total protein, were boiled for 5 minutes and separated by 6%SDS PAGE for 2 hours at 80 volts. Gels were blotted onto nitrocellulosemembranes using a semi-dry apparatus (BioRad). All membranes weresubsequently blocked (1 hour at room temperature) with 5% non-fat drymilk in PBS, 0.1% Tween 20 and incubated in primary antibodiesovernight at 4°C. Antibody conditions were as follows, all diluted inblocking buffer: primary antibody anti-C-cadherin (Developmental StudyHybridoma Bank, 6B6) 1:500 with secondary antibody GaM-HRP

(Jackson ImmunoResearch) 1:2500; primary antibody anti-human �-catenin (BD Transduction laboratory, C26220) 1:500 with secondaryantibody GaM-HRP 1:2500; primary antibody anti-�-catenin (Sigma,C2206) 1:2000 with GaR-HRP (Jackson ImmunoResearch) 1:2500;primary antibody anti-p120 (rabbit polyclonal) (Fang et al., 2004) 1: 5000with GaRb-HRP (Jackson ImmunoResearch) 1:2500. Membranesincubated with HRP-conjugated secondary antibodies were detected withECL developing solution (Amersham) and exposed to X-ray films(Hyperfilm, Amersham) at variable times to obtain unsaturated bands.

RESULTSC-cadherin is expressed as discrete punctae on thesurface of each blastomereC-cadherin is expressed on the surface membranes of blastomeresduring the cleavage and blastula stages (Choi et al., 1990; Levi etal., 1991). Cadherin clusters have been reported to be present aspunctae in the membranes of epithelial cells during their initialcontact, before becoming resolved into adherens junctions(Adams et al., 1998; Adams et al., 1996; Vasioukhin et al., 2000;Yonemura et al., 1995). To analyze the pattern of expression of C-cadherin in Xenopus blastulae, we followed the method describedin Kofron et al. (Kofron et al., 2002) (see also Fig. 1A). Dissected

2653RESEARCH ARTICLECortical actin assembly in Xenopus

Fig. 1. Expression of C-cadherin and its colocalization with cortical actin skeleton at the late blastula stage in Xenopus. (A) The assaysystem used in these experiments. (B) TCA-fixed animal caps were stained with an anti-C-cadherin monoclonal antibody (6B6) and visualized byCy5-conjugated goat anti-mouse IgG. Shown here is an optical slice of a cleared animal cap imaged by LSM confocal microscope. Positive stainingappears along cell-cell contacts as discrete punctae. (C) A grazing optical section in the plane of a cell membrane shows punctae en face (arrows).(D) Cells disaggregated in calcium- and magnesium-free saline lose surface cadherin. (E) Upon addition of calcium and magnesium, cells start toreaggregate. Two aggregating cells are shown at different angles. The membranes lining the site of initial adhesion between the cells producepunctae containing C-cadherin. (F) Cells on the blastocoelic surface of the animal cap stained with monoclonal antibody 6B6 against C-cadherin(red) and phalloidin. The two images are merged in the lower-left panel. Yellow areas indicate colocalization of F-actin and C-cadherin. (G) Theareas outlined in the merged image shown at high magnification. Arrows highlight colocalization of F-actin and C-cadherin. Scale bars: 20 �m inA-E; 10 �m in F,G.

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animal caps from late blastulae (Stage 9) were fixed for 30minutes in 2% trichloroacetic acid and stained as whole-mountswith a monoclonal antibody raised against C-cadherin (6B6)(Choi et al., 1990). Animal caps were cleared in Murray’s Clearand examined in a series of optical slices, starting at theblastocoelic surface. When individual 1-1.5 �m optical sliceswere assembled into stacks of 30-70, C-cadherin appeared toextend in a continuous line around each adjacent cell surface, aspreviously seen in conventional histological sections (Heasman etal., 1994). However, when individual 1.2 �m optical slices wereexamined, C-cadherin could be seen as a series of punctae aroundadjacent plasma membranes (Fig. 1B). Individual punctae weremeasured by pixel counting and had areas of 0.7±0.3 �m2 spacedby distances of 1.9±0.99 �m. This can be seen particularly clearlyin grazing sections through cell surfaces (arrows in Fig. 1C).When animal caps were dissociated in calcium- and magnesium-free buffered saline, C-cadherin was lost from the cell surfaces(Fig. 1D) (see also Hausen and Riebesell, 2002). When cells werereturned to calcium- and magnesium-containing saline andallowed to contact each other, punctae of C-cadherin rapidlyassembled at the sites of membrane apposition (Fig. 1E).

The cortical actin skeleton associates withcadherin punctaeSince we have shown previously that dissociated blastomeres lose thedense cortical actin network after cell dissociation and reassemble itwhen they are allowed to reassociate (Lloyd et al., 2005), thissuggested the hypothesis that the dense actin network might assembleon punctae of C-cadherin. To test this hypothesis, we fixed animalcaps from late blastulae in formaldehyde-glutaraldehyde fixative (seeMaterials and methods), stained them with Alexa 488-coupledphalloidin to reveal the cortical actin network, and then stained them

as whole-mounts with anti-C-cadherin antibodies. Fig. 1F,G showcortical actin filament bundles associated with C-cadherin-containingpunctae (arrows), interspersed with regions of cadherin-containingmembrane not associated with actin.

C-cadherin levels control the dense, but not thesparse, cortical actin networks in XenopusblastulaeThese data suggested that C-cadherin-containing complexesmight be required for cortical actin assembly at the membranes inXenopus blastulae, and that the pattern and density of corticalactin in the embryo could be controlled by the expression of C-cadherin itself, in addition to other possible levels of control. Totest this, we first depleted the maternal mRNA encoding C-cadherin, by injecting an antisense deoxyoligonucleotide (oligo)complementary to part of this mRNA, into cultured full-grownoocytes as described by Heasman et al. (Heasman et al., 1994).The oocytes were matured in vitro using 1 �M progesterone andfertilized by the host transfer method (Heasman et al., 1991). Fig.2A,B (left panels) show C-cadherin protein levels on the cellsurfaces in control and C-cadherin mRNA-depleted embryos atthe late blastula stage. There is a dramatic reduction in the levelof staining, consistent with previous findings (Heasman et al.,1994). The density of the cortical actin network wascorrespondingly decreased in the C-cadherin-depleted embryos(right panels of Fig. 2A,B). The normal, dense actin networkcharacteristic of interphase blastomeres was replaced by a sparsernetwork similar to that seen in dividing or dissociated cells.Second, C-cadherin mRNA (250-1000 pg per oocyte) wasinjected into oocytes before maturation. These were matured invitro and fertilized as above and the affects on cortical actinassembly examined at late blastula stage. Fig. 2C shows that the

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Fig. 2. The density of cortical actin dependsupon the level of expression of C-cadherin atthe cell surface. (A-C) Pairs of images fromdouble-stained (C-cadherin, red; F-actin, green)animal caps. (A) Control levels of C-cadherin andcortical actin. (B) An animal cap from a Xenopusembryo depleted of C-cadherin by antisense oligoinjection into oocytes. (C) An animal cap from anembryo overexpressing C-cadherin. Cortical actinis increased when C-cadherin is overexpressedand decreased when C-cadherin is depleted.(D) Quantitation of the cortical actin bymeasurement of pixel intensity. Scale bars:10 �m.

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level of C-cadherin staining (left panel) and the density of corticalactin (right panel) were both increased in C-cadherin-overexpressing animal caps compared with the controls (Fig. 2A).Pixel intensities from 5-10 caps were measured using the ZeissLSM 510 software. Statistically significant quantitative changesin actin staining confirmed the results seen in the individualimages (Fig. 2D). These data show that the dense actin network iscadherin-dependent, whereas the sparse actin network seen individing or dissociated cells is cadherin-independent.

The juxtamembrane region is required for C-cadherin to assemble a dense cortical actin networkThe function of transmembrane cadherins is regulated by theircytoplasmic domains, which bind a number of cytoplasmicproteins (Pokutta and Weis, 2002). Two major functional domainsof the cytoplasmic tail are the juxtamembrane region (JMR) andthe distal �-catenin/plakoglobin (�-catenin)-binding domain(CBD). We used mutant forms of C-cadherin to test the roles ofeach of these domains in actin assembly. First, we injected mRNAencoding a mutant Xenopus C-cadherin in which the residuesGGG (aa 731-733) in the JMR were replaced with AAA. Thismutant form is unable to bind p120 catenin (Thoreson et al.,2000). Unlike wild-type C-cadherin, overexpression of thismutant form did not increase the density of the cortical actin (Fig.3A, middle lower panel); in fact, it consistently decreased it (Fig.3B). A mutant form lacking the distal catenin-binding domain (C-cad �CBD, deletion of aa 839-896) did not significantly alter thedensity of cortical actin (Fig. 3A, right lower panel, Fig. 3B).

Immunostaining shows that both mRNAs were expressed (Fig.3A, upper panels). These data suggest that both the JMR and CBDdomains are required for C-cadherin to assemble the dense actinnetwork.

In this experiment, the endogenous cadherin was still presentand might have affected trafficking or presentation of the mutantcadherins. To control for this, we injected the two mutant mRNAsinto embryos whose endogenous C-cadherin had been depleted byantisense oligo injection into the oocytes. Fig. 4A shows thatC-cadherin mRNA depletion dramatically reduced the levelof cadherin protein on the cell surface. Both mutant formsof cadherin were efficiently translated, as shown byimmunocytochemical staining with anti-C-cadherin antibody. C-cad G-A was expressed on the cell surface and rescued celladhesion (Fig. 4A). However, it did not rescue the cortical actinnetwork. The only sites of actin polymerization were in densemembrane projections at cell boundaries (arrowed in Fig. 4B).However, over the rest of the cell cortex, there was no densecortical actin network in these cells. C-cad �CBD was notexpressed at the cell surface, but instead accumulated in largecytoplasmic vesicles (Fig. 4A,B). This explained the fact that itdid not rescue cell adhesion and precluded an assessment of thefunction of this mutant in actin assembly. Actin-rich cell-surfaceprojections were also seen in these cells, but there was no rescueof the cortical actin network over the rest of the cell surfaces. Theoverall levels of polymerized actin were quantitated by pixelintensity, as shown in Fig. 4C. Neither of the mutant forms ofcadherin rescued the loss of polymerized cortical actin caused by

2655RESEARCH ARTICLECortical actin assembly in Xenopus

Fig. 3. Effects of C-cadherin mutants on corticalactin assembly. (A) Animal caps dissected fromXenopus embryos at stage 9 were fixed with 2%TCA for anti-C-cad immunostaining (upper row) orwith FG fixative for F-actin staining (lower row).Pairs of images are shown from embryos that wereuntreated (Control, left), or injected with C-cad G-A(does not bind p120 catenin, center) or with C-cad�CBD (does not bind �-catenin, right). Bothcadherin mutant mRNAs are expressed (see also Fig.4). However, neither causes a significant increase incortical actin, and the C-cad G-A mutant causedloss of cortical actin. (B) F-actin levels quantitatedby pixel intensity. *, P<0.05. Scale bars: 20 �m.

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C-cadherin depletion. These data show that the p120-binding siteof C-cadherin is required for C-cadherin to assemble a densecortical actin network.

p120 catenin is required for dense cortical actinassemblyThe requirement for the JMR of C-cadherin in cortical actinassembly suggested that p120 catenin might be required for thisfunction. P120 catenin is expressed both maternally and zygoticallyin Xenopus. To test its role in cortical actin assembly at the blastulastage, a morpholino oligo (MO), previously shown to block thetranslation of p120 catenin mRNA (Fang et al., 2004), was injectedinto cultured Xenopus oocytes (10-40 ng per oocyte), which weresubsequently matured in vitro and fertilized. The amount of p120catenin protein was assessed by western blotting and found to bereduced in a dose-dependent manner by the mid-blastula stage (Fig.5B). The density of the cortical actin network was also significantlyreduced by p120 catenin depletion (Fig. 5A,C). This experiment wasrepeated with an antisense deoxynucleotide oligo, complementaryto a different region of the target mRNA (AS1, 7-9 ng per oocyte),which had the same effect (data not shown). The effects of both theMO and the antisense oligo were rescued by reintroducing p120catenin mRNA (shown for the antisense oligo experiment in Fig.5C), showing that in both cases, the effects were specific for p120catenin depletion. Thus, p120 catenin is required for cortical actinassembly at the blastula stage.

Next, synthetic p120 catenin mRNA in doses of 250 to 1000 pgwas injected into oocytes and the cortical actin examined at the lateblastula stage. Fig. 5A,D show that the density of the cortical actinnetwork was increased by the overexpression of p120 catenin. Weconclude that p120 catenin is both necessary and sufficient togenerate a dense cortical actin network.

p120 catenin is thought to regulate the steady-state levels ofcadherins on the cell surface (Chen et al., 2003; Davis et al., 2003; Eliaet al., 2006; Ireton et al., 2002; Perez-Moreno et al., 2006; Xiao et al.,2003). We therefore asked whether p120 catenin controls cortical actinassembly by controlling the level of C-cadherin on the surface of eachblastomere. Animal caps from p120 catenin-depleted blastulae werestained with 6B6 monoclonal antibody against Xenopus C-cadherin.Fig. 5E shows that depletion of p120 catenin caused a significantreduction in C-cadherin on the cell surface at the late blastula stageand, conversely, that injection of p120 catenin mRNA into the earlyembryo caused increased levels of C-cadherin at the cell surface (Fig.5F). These data show that p120 catenin plays an essential role incontrolling the steady levels of C-cadherin in the Xenopus embryoand, through this, controls the level of cortical actin assembly.

To confirm that interaction between C-cadherin and p120 cateninis required for their function in controlling the dense actin assembly,we generated a p120 catenin mutant that lacks the cadherin-bindingdomain (spanning the first Arm repeat, p120 �Arm1). The first Armrepeat has been shown to be necessary to target p120 catenin tocadherin on the cell membrane, but not to affect p120 cateninregulation of Rho GTPases (Anastasiadis et al., 2000; Yanagisawaet al., 2004). Overexpression of this construct did not increase eitherthe density of cortical actin or the level of C-cadherin on the cellsurface in Xenopus embryos (Fig. 5G,H), showing that the densecortical actin assembly requires interaction between p120 cateninand C-cadherin.

LPA and Xflop signaling control actin assembly bycontrolling the amount of C-cadherin expressionWe have shown previously that the dense actin network assembly inthe Xenopus blastula is controlled by two maternally expressed G-protein-coupled receptors, LPA1 and Xflop (Lloyd et al., 2005; Tao et

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Fig. 4. The C-cad G-A mutantdoes not rescue dense actinassembly when endogenousC-cadherin is depleted.(A) When overexpressed, C-cadG-A was localized on the cellsurface, but the C-cad �CBDmutant was trapped in theintracellular vesicles in Xenopusembryos depleted of C-cadherin.(B) F-actin (green) and C-cadherin (red) double stainingshows that the C-cad G-Amutant did not rescue the denseactin assembly. Arrows denoteactin-rich membrane processes.(C) Overall levels of polymerizedactin quantitated by pixelintensity. *, P<0.05. Scale bars:50 �m in A; 20 �m in B.

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al., 2005). We have shown above that the C-cadherin–p120 catenincomplex is a site of control of the dense cortical actin network. Wetherefore explored the possibility that one mechanism of Xflop andLPA function might be through regulation of C-cadherin levels. First,mRNAs encoding LPA1 (400 pg per embryo) or Xflop (250 pg perembryo) were injected into the animal cytoplasm at the 2-cell stage,and the embryos allowed to develop to the late blastula stage beforefixation and staining for C-cadherin. Fig. 6A-C show that bothmRNAs caused upregulation of C-cadherin levels. To distinguishbetween increased total protein and altered cellular distribution, weassayed the total C-cadherin levels by western blotting. Fig. 6D showsthat LPA or/and Xflop overexpression increased the total level of C-cadherin at the late blastula stage.

We next considered whether the two GPCRs could be acting asstructural components of the cadherin complex, rather than signalingthrough G proteins. Since the ligand for the LPA receptors, LPA,causes an increase in cortical actin (Lloyd et al., 2005), signal

transduction must be occurring. However, this has not been shownfor Xflop, as its ligand(s) is unknown. We therefore created the pointmutation R112A, which has been shown to block the activation ofG proteins by the motif of aa 111-113 (DRF) in the secondintracellular loop of GPCRs (Rovati et al., 2007), mRNA encodingXflop R112A was injected into each cell of the 2-cell embryo in awide range of concentrations, up to 1 ng. In no case did it mimic thewild-type Xflop in increasing the level of cortical actin (Fig. 7).These data confirm that signaling through the LPA and Xflopreceptors controls the level of C-cadherin at the late blastula stage.

The effects of LPA receptor and Xflop depletionon the dense cortical actin network can berescued by the overexpression of C-cadherinIf the LPA and Xflop signaling pathways control cortical actinassembly by regulating the amount of C-cadherin on the cell surface,then overexpression of C-cadherin should rescue the depletion of

2657RESEARCH ARTICLECortical actin assembly in Xenopus

Fig. 5. p120 catenin expression levelscontrol the level of C-cadherinexpression at the cell surface anddensity of the cortical actin skeleton.(A) Representative images of animal capsfrom Xenopus embryos that wereuntreated (Control), depleted of p120(p120 MO), or overexpressing p120 (p120RNA). (B) Western blot showing the degreeto which p120 protein levels are reduced atthe blastula stage. (C,D) The changinglevels of cortical actin, quantitated by pixelintensity, caused by p120 depletion by anmRNA-targeting antisensedeoxynucleiotide oligo (AS1) and rescue byp120 mRNA (C) and increasing doses ofp120 mRNA (D). *, P<0.05. (E) Imagesfrom C-cadherin-stained animal caps fromuntreated embryos (control) or p120-depleted embryos (p120 MO). (F) Imagesfrom untreated embryos (control) andembryos overexpressing p120 (p120 RNA).Depletion and augmentation of p120cause decrease and increase in the level ofC-cadherin at the cell surface, respectively.(G) A p120 mutant (p120 �Arm1) thatlacks the C-cadherin-binding domain hasno effect on either cadherin or corticalactin levels. C-cadherin (upper panels,samples were fixed with 2%TCA) and actin(lower panels, samples were fixed with FG)staining are shown for animal caps fromuntreated (left panels) and p120 �Arm1-injected embryos (right panels). Noincrease in C-cadherin or F-actin staining isseen. (H) Overall levels of F-actin caused byp120 �Arm1 overexpression quantitatedby pixel intensity. Scale bars: 10 �m in A;20 �m in E-G.

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these receptors. We therefore injected 500 pg of C-cadherin mRNAinto oocytes depleted of either LPA1 or Xflop by injection ofantisense oligos into cultured oocytes. In both cases, the densecortical actin network was restored (Fig. 8B-E). Depletion of eitherXflop or LPA reduced both the membrane (Fig. 8A,B,C) and total(Fig. 8D,E insets) levels of C-cadherin, further supporting the notionthat Xflop and LPA receptors control the level of C-cadherin at thelate blastula stage in Xenopus.

C-cadherin is necessary for Xflop to control denseactin assemblyAs shown above, signaling through both Xflop and LPA receptorscontrols the amount of cadherin on the cell surface, on which actinis polymerized. Actin filaments can be assembled on a number ofdifferent cell-surface proteins and so it is possible that thesereceptors also control actin assembly on other surface proteins. If so,overexpression of the receptors would rescue cortical actin in theabsence of C-cadherin. To test this, we depleted C-cadherin usingantisense oligos injected into cultured oocytes, as above, andinjected mRNAs encoding either Xflop or LPA1 at the 2-cell stage.Xflop overexpression (200 pg mRNA) did not rescue the densecortical actin network in the absence of cadherin, although it didincrease the cortical actin density in control embryos (Fig. 9),suggesting that it acts only through the cadherin complex. LPA1overexpression did increase cortical actin density after depletion ofC-cadherin (not shown). This confirms a previous finding that the

addition of the LPA ligand to dissociated cells in culture, which donot express cadherins, led to increased actin assembly (Lloyd et al.,2005). This suggest that LPA signaling, at least at higher thanphysiological levels, can control the assembly of actin by othermechanisms in addition to its role in cadherin expression.

DISCUSSIONPrevious work has shown that the cortical actin skeleton, in additionto its known roles in the movements of gastrulation and incytokinesis, also provides a skeletal framework for the embryos atthe blastula stage. Abrogation of the cortical actin skeleton at the lateblastula stage causes collapse of the entire embryo and failure ofgastrulation movements (Kofron et al., 2002). It is therefore of greatinterest to understand the mechanism of cortical actin assembly inthe early embryo. Previous work also showed that dissociated cells,in which cell contact and signaling are prevented, lose their densecortical actin, and that the G-protein-coupled receptors LPA1 andLPA2, and Xflop, are required for cortical actin assembly in vivo(Lloyd et al., 2005; Tao et al., 2005). However, the mechanism ofaction of these receptors, and whether other aspects of cell contactare required, have not been explored. Here, we show that there is adirect relationship between the expression of C-cadherin on the cellsurface and the assembly of cortical actin in the cytoplasm, and thatthe level of cell-surface cadherin is controlled by signaling throughGPCRs. This interacting system results in the level of cell cohesionand rigidity seen at the late blastula stage.

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Fig. 6. Gain-of-function experimentsshow that the G-protein-coupledreceptors Xflop and LPA2 control thelevel of C-cadherin on the cellsurface. (A,B) Cadherin-stained animalcaps from untreated animal caps(Control) and animal caps from Xenopusembryos injected with Xflop (A, rightpanel) or LPA2 (B, right panel) mRNA.(C) Quantitation of pixel intensityshowing that both mRNAs increased thelevel of cadherin staining. *, P<0.05.(D) Western blot showing the increase inthe total levels of C-cadherin caused byoverexpression of Xflop and LPA mRNAs.-tubulin was used as loading control.Scale bars: 20 �m.

Fig. 7. Overexpression of an Xflop mutant (R112A) that lacks G-protein-coupling activity does not increase cortical actin assembly.(A) Alexa 488-conjugated phalloidin staining showing that overexpression of the R112A mutant (1 ng mRNA injected) does not mimic the capacityof wild-type Xflop to increase cortical actin assembly. (B) Quantitation by pixel intensity shows that R112A had no effect on cortical actin assembly.*, P<0.01. Scale bar: 50 �m.

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It is already known that cadherin-containing punctae form at sites ofinitial contact of epithelial cells in culture (Helwani et al., 2004;Kobielak et al., 2004; Kovacs et al., 2002b; Scott et al., 2006;Vasioukhin et al., 2000; Verma et al., 2004), and that these associatewith actin filaments and actin assembly components such as Arp2/3(Kovacs et al., 2002b; Verma et al., 2004), formin 1 (Kobielak et al.,

2004) and Ena/Vasp (Scott et al., 2006). Here we show, in a developingsystem in vivo, that such punctae and their associated actin filamentassemblies form the skeleton of the early embryo. The presentation ofC-cadherin on the cell surface is clearly a rate-limiting step of thisprocess, as its overexpression increases the dense cortical actinnetwork, whereas its depletion causes loss of the dense cortical actin.

2659RESEARCH ARTICLECortical actin assembly in Xenopus

Fig. 8. Depletion of Xflop or LPAreceptors causes reduced C-cadherin expression and reducedcortical actin assembly that canbe rescued by C-cadherin mRNA.(A-C�) Pairs of images (left, C-cadherin staining; right, F-actinstaining) from Xenopus embryosthat were untreated (A), Xflop-depleted (B), or LPA1 and LPA2-depleted (C). Both cadherin stainingand cortical actin assembly arereduced by Xflop and LPA1/2depletion. The effects of eachdepletion can be rescued bysubsequent injection of C-cadherinmRNA (B�,C�). (D,E) Cortical actinlevels were quantitated by pixelintensity. *, P<0.01. Insets arewestern blots showing thatdepletion of Xflop (D) or LPA1/2 (E)receptors also reduced the totallevels of C-cadherin. tubulin wasused as a loading control. Scalebars: 10 �m.

Fig. 9. Depletion of C-cadherin blocks Xflop-induced cortical actin assembly. (A) Alexa 488-conjugated phalloidin staining of F-actin shows that C-caderin depletion (c-cad depl) significantly reduces bothendogenous and Xflop overexpression induced corticalactin assembly. (B) This notion is supported by pixelintensity quantitation. *, P<0.05. Scale bar: 50 �m.

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The importance of C-cadherin for dense cortical actin assemblyis supported by the finding that p120 catenin was also found to beboth necessary and sufficient for this process. p120 catenin is knownto be important for stabilizing cadherin on the cell surface (Davisand Reynolds, 2006; Elia et al., 2006; Perez-Moreno et al., 2006)and this proved to be the case here. p120 overexpression causedincreased amounts of both C-cadherin and cortical actin, whereasdepletion caused the reverse. In addition, a p120 mutant lacking thecadherin-binding site failed to increase either C-cadherin expressionon the surface, or cortical actin assembly, showing that the level ofC-cadherin expression on the cell surface requires its interactionwith p120. It is possible that p120 controls cortical actin assemblyat other levels, in addition to cadherin presentation. Our data suggestthat p120-mediated cortical actin assembly requires its interactionwith C-cadherin. The fact that a cadherin construct lacking the p120-binding site was expressed at high levels on the cell surface, butcaused loss of cortical actin filaments, supports this hypothesis. p120catenin has been shown to bind signaling intermediates in actinassembly such as Rac1 and Cdc42 (Noren et al., 2000), so it will beinteresting to see whether these interactions are also required foractin assembly in Xenopus. A cadherin construct lacking the �-catenin-binding domain and that does not bind �-catenin (Pokuttaand Weis, 2002) failed to significantly increase cortical actinassembly. However, in the absence of endogenous C-cadherin, thisconstruct was not transported to the cell membrane and did notrescue cell adhesion. The role of the CBD in cortical actin assemblycould therefore not be tested with this mutant. However, ourobservations do suggest a potential role for the CBD in proteintrafficking and membrane presentation of cadherin. The high levelof membrane expression of the cadherin mutant lacking the p120-binding site might also be due to the intact CBD in this mutant.

Once it became clear that the late blastula cells somehow titratethe assembly of cortical actin against the level of C-cadherin on thesurface, this immediately raised the novel possibility thatintercellular signaling through GPCRs might control the density andpattern of cortical actin in the blastula at the level of cadherinexpression. This indeed proved to be the case. Depletion oraugmentation of the receptors LPA1 and LPA 2, and Xflop,previously shown to be required for cortical actin assembly (Lloydet al., 2005; Tao et al., 2005), caused corresponding loss and increaseof C-cadherin expression on the cell surface. Furthermore,overexpression of C-cadherin by mRNA injection was able to rescuecortical actin assembly after depletion of either LPA1 or Xflop in theblastula. These data implicate the two signaling pathways incontrolling the level of expression of cadherin on the cell surface. Itwill be of major interest to discover whether these signalingpathways are the sites of action of cell cycle components, as thedense cortical actin network is lost during cell division.

One interesting and unexplained fact in these studies is that thereappears to be two actin-containing networks in the blastula cells.Cells that are dividing in vivo, or dissociated in calcium- andmagnesium-free saline, or depleted of C-cadherin, all lose theirdense cortical actin network, but retain a sparse network, even in theabsence of cadherin on the cell surface (Fig. 2) (see also Lloyd et al.,2005). This sparse network is clearly cadherin-independent and isindependent of the dense network. We do not know if the reverse isthe case, as we have not found any independent way of removing thesparse network. It is possible the LPA and Xflop signaling alsocontrol the sparse network, although experimental details are stilllacking. We also do not know how the sparse network is associatedwith the cell cortex. There are several known sites of actin insertioninto the cell membrane, in addition to cadherins. For example,

-spectrin and moesin, members of FERM (band 4.1/ezrin/radixin/moesin) proteins are maternally expressed in Xenopusoocytes (Carotenuto et al., 2000; Thorn et al., 1999), and could playa role in attaching the sparse actin network to the cell membrane.Some integrins (�1, for example) are present at the blastula stage(Gawantka et al., 1992) and could be sites of actin assembly.

Many more questions remain. We do not know the mechanism bywhich LPA and Xflop signaling control the level of C-cadherin onthe cell surface. It is also possible that LPA and Xflop control actinassembly at the cadherin complex at levels other than via C-cadherinexpression. They might also control the sparse network throughmechanisms that do not involve cadherin expression. The Xenopusblastula represents a simple in vivo system with which to addressthese questions.

This work was supported by Cincinnati Children’s Hospital ResearchFoundation and the National Institutes of Health (RO1-HD044764). Theauthors declare that they have no competing financial interests.

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