Planar Cell Polarity Genes Regulate Polarized Extracellular Matrix Deposition during Frog Gastrulation

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1 Current Biology, Vol. 15, , April 26, 2005, 2005 Elsevier Ltd All rights reserved. DOI /j.cub Planar Cell Polarity Genes Regulate Polarized Extracellular Matrix Deposition during Frog Gastrulation Toshiyasu Goto, 1,2,5 Lance Davidson, 2,3,5 terior body axis. Convergence and extension movements Makoto Asashima, 1,4 and Ray Keller 2, * are the result of several rounds of mediolaterally 1 International Cooperative Research Project directed cell intercalation. In mesodermal tissues, med- Japan Science and Technology Corporation iolateral intercalation coincides with characteristic bi- Japan polar filo-lamelliform protrusive activity directed along 2 Department of Biology the mediolateral axis called mediolateral cell intercala- University of Virginia tion behavior (MIB) [3]. Overexpression, expression of Charlottesville, Virginia dominant-negative forms, or morpholino-mediated 3 Department of Cell Biology knockdowns of components of the PCP pathway, in- University of Virginia School of Medicine cluding Disheveled [4], Strabismus (Van Gogh) [5] (Fig- Charlottesville, Virginia ure 1A), Frizzled [6, 7], Prickle [8], and Wnt 11 [9], as 4 Department of Life Sciences (Biology) well as downstream signaling components, including a Graduate School of Arts and Sciences cytoplasmic formin homology protein DAAM1, and the The University of Tokyo small rho family GTPases, Rho, Rac, and Cdc42 [ Komaba, Meguro-ku 13], result in failure of convergent extension, failure of Tokyo blastopore closure in most cases, and produce a very Japan wide but severely shortened anterior-posterior axis. Several findings raise the question of whether the role of PCP pathway genes in MIB and the resulting Summary convergent extension is direct, indirect, or both and suggest an indirect role in organizing extracellular matrix The noncanonical wnt/planar cell polarity (PCP) pathtracellular (ECM). Fibronectin, a principle component of the ex- way [1] regulates the mediolaterally (planarly) polarmajor matrix and its integrin receptor, α 5 β 1, plays a ized cell protrusive activity and intercalation that role in radially polarizing animal cap and marginal drives the convergent extension movements of vertethat zone cells, a role essential for the radial intercalation brate gastrulation [2], yet the underlying mechanism drives epiboly and early extension of the marginal is unknown. We report that perturbing expression of zone of Xenopus [14]. In addition, an integrin-mediated Xenopus PCP genes, Strabismus (Xstbm), Frizzled interaction with fibronectin regulates cadherin-depen- (Xfz7), and Prickle (Xpk), disrupts radially polarized dent adhesion and is essential for the subsequent medfibronectin fibril assembly on mesodermal tissue surdrives convergent extension [15]. Perturbation of Wnt iolateral intercalation of deep mesodermal cells that faces, mediolaterally polarized motility, and intercalasignaling and noncanonical Dsh (Dishevelled) function tion. Polarized motility is restored in Xpk-perturbed explants but not in Xstbm- or Xfz7-perturbed explants disrupts organized fibrillar matrix deposition in Xeno- pus and blocks cell polarization and convergent extencultured on fibronectin surfaces. The PCP complex, sion (Marsden and DeSimone, 2003, 43rd Annu. Meet. including Xpk, first regulates polarized surface as- Am. Soc. Cell Biol., abstract). The function of the small sembly of the fibronectin matrix, which is necessary GTPases Rho and Rac [16] are essential for extracellufor mediolaterally polarized motility, and then, without lar matrix fibril organization by cultured cells in vitro, Xpk, has an additional and necessary function in posuggesting that these genes, which are downstream of larizing motility. These results show that the PCP the PCP pathway [10, 11], may mediate PCP function complex regulates several cell polarities (radial, plain matrix deposition. nar) and several processes (matrix deposition, motil- In normal development of Xenopus, fibronectin (FN) ity), by indirect and direct mechanisms, and acts in is a major component of the fibrillar ECM that lines the several modes, either with all or a subset of its comblastocoel roof and surrounds the mesoderm [17] durponents, during vertebrate morphogenesis. ing convergence and extension (Figures 1B and 1C, Results and Discussion controls). In fibronectin fibril assembly, overexpression of Xstbm mrna produces dose-dependent defects (Figures 1B and 1C), which are correlated with worsen- Vertebrate homologs of the Drosophila genes function- ing whole-embryo defects in convergent extension ing in the PCP pathway are necessary for the polarized from weak to mild to severe (Figure 1A). Fibrils are not cell behaviors underlying the convergent extension simply absent but are instead assembled in a disorgamovements that function in gastrulation and shape the nized manner in both the planar, enface projection (Figbody plan [2]. In frog embryos, the axial and paraxial ure 1B) as well as across the thickness of the mesomesodermal tissues and the neural tissues narrow derm as seen in the transverse projection (Figure 1C). (converge) and lengthen (extend) dramatically during Notably, transverse projections show progressive failgastrulation and neurulation, which results in squeezing ure to localize fibril deposition to the external surfaces the blastopore closed and elongating the anterior-pos- of the mesodermal tissue masses (Figure 1C). Similar fibril assembly defects are found in whole embryos exhibiting *Correspondence: rek3k@virginia.edu severe phenotypes after overexpression of 5 These authors contributed equally to this work. Xstbm, Xfz, Xpk, and the dominant-negative constructs

2 Current Biology 788 Figure 1. Whole-Embryo Phenotypes and Fibronectin Fibril Distribution in the Dorsal Axis (A) Increasing severity of whole-embryo phenotype with amount of Xstbm overexpression. (B and C) Increasing disruption of fibrillar fibronectin ECM organization with increasing amounts of Xstbm overexpression. (B) Projection of en face confocal sections (shown in schematic at left) shows increasing width of notochord field (asterisk) and uneven lateral boundaries with somitic mesoderm (arrowheads). (C) Projection of transverse sections (shown in schematic at left) shows that increasing amounts of Xstbm result in increasing amounts of fibril assembly throughout the notochord (asterisk) no longer restricted laterally by the somitic mesoderm (arrowheads). (D F) Expression of dominant-negative Xstbm PDZ-B and Xpk Lim-PET and overexpression of Xfz7 and Xpk produce severe whole-embryo phenotypes similar to severe Xstbm phenotypes (D), disrupt both planar (E) (en face projections of confocal z sections) and radial organization (F) (transverse projection of confocal z sections) of fibronectin ECM throughout the dorsal axial mesoderm (asterisk, notochord; arrowhead, lateral boundary of notochord and somitic mesoderm). Scalebars indicate 50 microns. Scale of (B) and (E) are the same, as are (C) and (F). ant, anterior; pos, posterior; dor, dorsal; ven, ventral; so, somitic mesoderm. directly responsible for the defects in convergent extension or whether fibril disorganization was a conse- quence of failed convergence and extension, we assessed the role of Xstbm, Xfz7, and Xpk on cell intercalation when an ectopic FN substrate was provided. Because Xenopus notochord cells normally express Xpk, Xstbm, and Xstbm dpdz-b and Xpk dlim-pet (whole-embryo phenotype, Figure 1D; enface FN organization, Figure 1E; transverse FN organization, Figure 1F). Thus, fibronectin disorganization and nonpolar assembly of fibrils increases with increasingly severe PCP phenotypes. To assess whether perturbed fibronectin fibrils were

3 PCP Genes Regulate ECM during Gastrulation 789 Xfz7 [5, 8, 18], we used the three-notochord explant rected into the mediolateral quadrants; Xfz7-overexpressing preparation (Figure 2A) in which three notochordal anlage explants are similar to Xstbm cells, data not are grafted together, side by side, with the center shown). and side notochords labeled differently; the cells of Our results argue that the PCP pathway has two these notochords normally intercalate mediolaterally to functions in the early development of the frog embryo. form one notochord and elongate (Figure 2B, control/ Its first and perhaps primary role is for the directed, BSA), thereby allowing direct observation of cell inter- polarized assembly of fibronectin fibrils along tissue interfaces calation. After 5 hr, control explants narrow by 47%, surrounding the mesoderm (e.g., the surface and by 10 hr, they have narrowed by 69%. In contrast, of the mesoderm; Figure 4). By polarized we mean by 10 hr Xpk, Xstbm, and Xfz7 explants narrow by 38%, the localized assembly of fibronectin along the surface 11%, and 14%, respectively (Figure 2B). In control ex- of the mesoderm. In regard to this role, perturbation of plants cultured on BSA-coated glass, cells adopt a the PCP pathway of the frog acts cell nonautonomously characteristic elongated bipolar shape (10.51 length/ and results in unpolarized (nonsurface) assembly of the width ratio), intercalate freely and deeply into the neighboring ECM. Polarized deposition of this fibronectin matrix is regions, and the explant extends, whereas in ex- necessary for the subsequent polarization of cell motil- plants overexpressing Xstbm, or Xfz7, the elongated bipolar ity in the mediolateral axis, and in its absence, this cell shape (both 1.48 L/W), cell intercalation, and characteristic mediolateral intercalation behavior (MIB) convergent extension do not occur (Figure 2B). Note does not occur. In Drosophila, Fz, Stbm, Dsh, and Pk that the labeled lateral notochords (bright) intercalate are thought to form a feedback complex that functions completely into the unlabeled medial notochord in to develop polarized distribution of proteins, with controls but not in the others (Figure 2B). Weak cell Fz and Dsh high on one end of the cell and Stbm and intercalation and convergence occurred in the Xpkoverexpressing Pk high on the other, thus polarizing the cell [20, 21]. explant; however, cells do not adopt The fact that perturbation of Xstbm, Xfz, and Xpk in this elongated bipolar shapes (1.49 L/W; Figure 2B). In contrast, study, and Wnt 11 and Dsh in another study (Marsden in Xpk-overexpressing explants cultured on fibro- and DeSimone, 2003, 43rd Annu. Meet. Am. Soc. Cell nectin-coated glass, the cells orient mediolaterally, Biol., abstract), all result in failure of polarized matrix adopting the bipolar, fusiform cell shape (4.42 L/W) at assembly argues that the PCP complex, including Xpk, a level approximating that seen in control explants (4.47 is essential for this first function of polarized assembly L/W) cultured on fibronectin-coated glass (Figure 2C). of matrix. Xpk may localize, or locally activate the PCP However, these features of mediolateral cell polarization complex in response to radial tissue polarity cues al- are not restored in explants overexpressing Xstbm ready present in the embryo and by unknown mecha- (1.48 L/W) or Xfz7 (1.52 L/W) cultured on fibronectincoated nisms, perhaps involving interaction with the integrin glass (Figure 2C). Note that explants cultured pathway, localize fibril deposition to the mesodermal on fibronectin substrate do not converge and extend surfaces (Figure 4). Xpk s role might be to act as a sim- substantially [19]. The fibronectin is attached to a rigid ple on/off switch, as it does in the Drosophila wing substrate, the glass coverslip, and the cells attached to disk [21, 22]. In that case, an intact PCP complex is FN therefore can not distort the coverslip. In the entrained to the proximal/distal polarity already established embryo, however, the matrix is fibrillar and deformable in the disk. In Xenopus, Xpk may act by localizing [17], thus allowing convergence and extension. the PCP complex to the surfaces of the mesoderm cell To rule out the possibility that disrupting PCP gene facing ectoderm or endoderm. expression reduced protrusive activity and confirm ef- The fact that failure of Xpk function, but not that of fects on mediolaterally polarized protrusive activity, we the other PCP genes, can be rescued by an exogenous characterized cell protrusions in dorsal open-faced ex- planar surface of fibronectin suggests a second, Xpkindependent plants expressing a membrane-targeted GFP (GAP43- function of the PCP complex in mediolatplants GFP). Explants made at stage 10 and cultured on fibro- eral cell polarization and suggests that assembly of a nectin-coated glass expose deep cells of the explant planar matrix is the essential function of first activity of to high resolution confocal time-lapse imaging at the the PCP complex and that exogenously provided polarized cell-substrate surface (red) and 5 m deep (green). Within matrix can substitute for the first, Pk-dependent 6 hr, both control (Figure 3A, green) and Xpk-overex- operation of the PCP complex (Figure 4). The fact that pressing cells (Figure 3C, green) adopt the bipolar perturbation of the other PCP genes cannot be rescued shape characteristic of MIB, whereas Xstbm-overex- suggests that they are essential for this second operation pressing cells remain round (Figure 3B, green). Control of the complex. and Xstbm- and Xpk-overexpressing cells extend and The polarized, surface assembly of matrix mediated retract lamellipodia (Figures 3A, 3B, and 3C, arrowheads). by the whole PCP complex may provide the radial The frequency of lamellipodial-protrusive activ- context cues that persist into the next, mediolateral po- ity was similar in all cases (30 per cell per hour in control larization phase of mesodermal morphogenesis. This cells, 37 in Xstbm-overexpressing cells, and 26 in persistent radial polarization cue from the first phase Xpk-overexpressing cells); however, protrusions were may be essential for the second activity phase of the randomly directed in Xstbm-overexpressing explants PCP complex minus Pk in mediolateral (planar) polarization. but polarized mediolaterally in both control and in Xpkoverexpressing The defining systems for the PCP pathway are explants (Figures 3A#, 3B#, and 3C#) the Drosophila wing and eye, both of which are epithelial (92% of protrusions from control cells, 57% of protrusions sheets with inherent apical-basal polarity [23], but from Xstbm-overexpressing cells, and 85% of the mesoderm in the frog, as in most vertebrates, is a protrusions from Xpk-overexpressing cells were di- mesenchymal mass of cells [24] with no inherent radial

4 Current Biology 790

5 PCP Genes Regulate ECM during Gastrulation 791 Figure 3. Cell Protrusions along the Fibronectin Substrate in Explants Overexpressing Xpk or Xstbm Xpk or Xstbm mrna was injected into two dorsal blastomeres of the 4-cell-stage embryo. An mrna encoding a plasma membrane localizing GFP (gap43-gfp) was injected into several dorsal blastomeres at stage 7 to allow visualization of cell protrusions and cell shapes. Dorsal open-faced explants were dissected from the injected embryos at stage 10 and cultured on the fibronectin-coated glass exposing the deep cells to confocal time-lapse imaging. The protrusions were detected on the surface on labeled cells facing the fibronectin substrate (red) and cell shapes were observed 5 m deeper into the explant (green). (A) In control explants, gap43-gfp-labeled cells adopt bipolar shapes with protrusions at their mediolateral ends. (B) Cells in Xstbm-overexpressing explants remain isodiametric, do not adopt bipolar shapes, and extend protrusions in all directions. (C) Cells in Xpk-overexpressing explants show similar bipolar shapes and mediolaterally directed protrusions. Arrowheads show lamelliform protrusions along the fibronectin substrate. (A#, B#, and C#) Analysis of protrusive activity in (A), (B), and (C), respectively. Rose diagrams show the normalized frequency of protrusions from five cells in the explant shown in (A), eight cells in the explant shown in (B), and seven cells shown in the explant shown in (C) directed into 30 bins representing 360 around the cell s center. axis (e.g., deep-superficial or apical-basal axis). Polarized matrix assembly in the first operation of the PCP complex may provide this essential radial axis, thereby allowing the PCP complex to respond to upstream or parallel global planar cues [25] in its second and more proximal role of planarly polarizing MIB. Figure 2. Cell Intercalation in Three-Notochord Explants (A) Procedure for making three-notochord explants. Xstbm, Xpk, or Xfz7 mrna with green tracer (GFP or gap43-gfp) or red tracer (Alexa594 dextran) was coinjected into two dorsal blastomeres of the 4-cell-stage embryo. Notochord sectors (without epithelium) were dissected from injected embryos at stage 10. (B and C) Three-notochord sectors from different embryos were grafted together and cultured on either the BSA (B)- or fibronectin (C)-coated glass, exposing the deep cells to two fluorescent color time-lapse imaging. Control explants were made from embryos injected with GFP or gap43-gfp and Alexa594 dextran. The time elapsed is indicated at the bottom right. (B) On BSA, cells in control explants adopt bipolar shapes and intercalate deeply into the neighbor notochord to make a single big notochord. Xpk-overexpressing explants converged weakly. Xstbm- and Xfz7-overexpressed cells remain round and the explants never converge. (C) On fibronectin, cells in the central notochord of control explants adopt bipolar shapes and intercalate deeply. Likewise, cells within Xpk-overexpressing explants become bipolar and intercalate. In contrast, Xstbm- and Xfz7-overexpressed cells remain round and do not intercalate as in (B). ml, mediolateral.

6 Current Biology 792 Figure 4. A Two-Step Model for Function of the PCP Complex in Cell Polarization during Convergent Extension Viewed in the Transverse Section The first step (blue arrow, center) involves radial polarization of the mesodermal cells (red shading) and the associated, polarized secretion of fibronectin extracellular matrix (green) at the outer surfaces of the mesodermal cells, between the mesoderm and the overlying neural (dark gray) and underlying endodermal (light gray) epithelial tissues. Tissue interactions not yet described between one or both of these epithelial tissues and the mesoderm (black arrows, center, top) may induce this radial polarization of the mesodermal cells. As a consequence, in a polarized fashion at their outer ends, the cells assemble a fibronectin matrix, which is at the interfaces between the mesenchymal mesoderm and these adjacent epithelial tissues (green, center). In the second step (magenta arrow, center), the cells polarize mediolaterally (red shading), enabling them to produce their characteristic mediolateral intercalation behavior and, thus, convergent extension. Perturbation of Prickle function on the one hand (Pk, center left) and Stbm or Fz on the other (Stbm, Fz, center right) result in failure (light blue arrows) of radial polarization and assembly of disorganized, unpolarized matrix (nonsurface; green). Subsequently, mediolateral polarization also fails, and the tissue thickens (because of failure of radial intercalation) and fails to converge and extend (light magenta arrows). Culture of Pk-perturbed explants on a planar, exogenous fibronectin substrate rescues mediolateral polarization (far left), whereas it does not rescue Stbm- or Fz-perturbed explants (far right). These results argue that the PCP complex, including Prickle, is necessary for the first step (blue arrows) of radial polarization of the cells and, directly or indirectly, for polarized, surface assembly of fibronectin matrix, but the second step (magenta arrows) requires the polarized, surface matrix and the PCP complex but not Prickle. (1000 pg), Xstbm ( pg), Xstbm ( PDZ-B) (500 pg), Xfz7 (100 pg), and GFP (5 200 pg) or gap43-gfp (5 200 pg) as a green tracer and 800 pg of Alexa594-dextran (Molecular Probes, Eugene, Oregon) as red tracer were microinjected into the prospective anterior marginal zone of the two dorsal blastomeres of the 4-cell embryo. Microsurgery, Imaging, and Morphometric Analysis Procedures for making three-notochord explants [5] and marginal- zone explants [19] were previously described. Glass was coated overnight with either 20 g/ml bovine fibronectin (Roche Molecular Biochemicals, Indianapolis, Indiana) or 1 mg/ml BSA (Sigma Chem- ical, St. Louis, Missouri). Low-light fluorescence images and time- lapse recordings of cell behavior were collected with a digital cam- era (Hamamatsu 4742, Orca, Bridgewater, New Jersey) mounted on an inverted compound microscope (Olympus IX70; Olympus, Melville, New York) controlled by image acquisition software (Metamorph Imaging System; Universal Imaging, Downington, Pennsyl- vania). Confocal time-lapse and z-series stacks of confocal optical sections were collected with 60 and 20 objectives, respectively, by using a confocal scanning laser microscope (PCM2000; Nikon, Melville, New York) mounted on an inverted compound microscope (Nikon TE-200) controlled by acquisition software (Compix, Cran- berry Township, Pennsylvania). Projections of z series to single images and assembly of two-level time-lapse sequences were carried out with image-processing software (ImageJ; Wayne Rasband, NIMH; see for ImageJ). Explant convergence, cell shapes, and protrusive activity were determined from time-lapse sequences and measured with quantitative-image-analysis software (Metamorph Imaging System and ImageJ). Rose diagrams of protrusive activity and cell-shape orien- tation were plotted with macros written by one of the authors (L.D.; NIH-Image, Wayne Rasband, NIMH; see nih-image). Acknowledgments We thank the members of the Keller Lab, Mungo Marsden, and Doug DeSimone for thoughtful comments and suggestions in the course of this work. Additionally, we would like to thank Jeff Miller These considerations highlight the fact that although the PCP pathway appears homologous in vertebrates and invertebrates (Drosophila) there are several impor- tant cell biological differences. First, vertebrate mesodermal cells at this stage are mesenchymal, not epithelial. In the case of the wing disk, the eye, and other epithelia of Drosophila, disruption of the primary genes in the PCP pathway still results in the generation of po- larized structures albeit misoriented ones [1]. In the case of the Xenopus mesoderm, cells are not simply misoriented but lose their polarity as well. Mediolateral cell intercalation depends on anterior-posterior positional information in both Xenopus [25] and in Drosophila [26], but in Drosophila, it does not appear to be regulated by the PCP pathway, suggesting that despite a common dependence on anterior-posterior signaling and a common mode of extension by cell intercalation, convergent extension in the two may be regulated differently. Lastly, in Drosophila epithelial tissues, the PCP pathway regulates development of a permanent structural polarity, whereas in frog mesoderm, the PCP path- way regulates a reiterative polarized cell behavior that produces several rounds of cell intercalation. Any mechanism of initiating polarity in vertebrate mesoderm must continue to operate continuously through multiple rounds of cell intercalation as well as during the long process of recruiting cells and organizing MIB in the posterior dorsal axis. Experimental Procedures Microinjection and Immunostaining Whole embryos were staged [27] and fixed with 5% trichloroacetic acid in phosphate-buffered saline and stained with monoclonal antibody (4H2) to Xenopus fibronectin [17]. mrnas encoding Xpk

7 PCP Genes Regulate ECM during Gastrulation 793 and Eddy DeRobertis for the GAP-43 plasmid, Peter Kline for the 19. Davidson, L.A., Keller, R., and DeSimone, D. (2004). Patterning Frizzled construct, and Ammasi Periasamy and the W.M. Keck Center for Cellular Imaging, University of Virginia. This work was sup- marginal zone of Xenopus laevis. Gene Expr. Patterns 4, 457 and tissue movements in a novel explant preparation of the ported by National Institutes of Health RO1 HD (to R.E.K.) 466. and a grant from the International Cooperative Research Project, 20. Jenny, A., Darken, R.S., Wilson, P.A., and Mlodzik, M. (2003). Japan Science and Technology Agency (to M.A.). Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. EMBO J. 22, Received: December 10, Tree, D.R., Shulman, J.M., Rousset, R., Scott, M.P., Gubb, D., Revised: February 28, 2005 and Axelrod, J.D. (2002). Prickle mediates feedback amplifica- Accepted: March 2, 2005 tion to generate asymmetric planar cell polarity signaling. Cell Published: April 26, , Bastock, R., Strutt, H., and Strutt, D. (2003). Strabismus is References asymmetrically localised and binds to Prickle and Dishevelled during Drosophila planar polarity patterning. Development 130, 1. Adler, P.N. (2002). Planar signaling and morphogenesis in Dro sophila. Dev. Cell 2, Knust, E. (2000). Control of epithelial cell shape and polarity. 2. Keller, R. (2002). Shaping the vertebrate body plan by polarized Curr. Opin. Genet. Dev. 10, embryonic cell movements. Science 298, Keller, R., Davidson, L.A., and Shook, D.R. (2003). How we are 3. Shih, J., and Keller, R. (1992). Cell motility driving mediolateral shaped: the biomechanics of gastrulation. Differentiation 71, intercalation in explants of Xenopus laevis. Development 116, Ninomiya, H., Elinson, R.P., and Winklbauer, R. (2004). Antero- 4. Wallingford, J.B., Rowning, B.A., Vogeli, K.M., Rothbacher, U., posterior tissue polarity links mesoderm convergent extension Fraser, S.E., and Harland, R.M. (2000). Dishevelled controls cell to axial patterning. Nature 430, polarity during Xenopus gastrulation. Nature 405, Zallen, J.A., and Wieschaus, E. (2004). Patterned gene expres- 5. Goto, T., and Keller, R. (2002). The planar cell polarity gene sion directs bipolar planar polarity in Drosophila. Dev. Cell 6, strabismus regulates convergence and extension and neural fold closure in Xenopus. Dev. Biol. 247, Nieuwkoop, P.D., and Faber, J. (1967). Normal Tables of Xeno- 6. Djiane, A., Riou, J., Umbhauer, M., Boucaut, J., and Shi, D. pus laevis (Daudin) (Amsterdam: Elsevier North-Holland Bio- (2000). Role of frizzled 7 in the regulation of convergent extenmedical Press). sion movements during gastrulation in Xenopus laevis. Development 127, Medina, A., Reintsch, W., and Steinbeisser, H. (2000). Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 92, Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, T.S., Takahashi, H., Takeda, H., and Ueno, N. (2003). The pricklerelated gene in vertebrates is essential for gastrulation cell movements. Curr. Biol. 13, Tada, M., and Smith, J.C. (2000). Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127, Habas, R., Dawid, I.B., and He, X. (2003). Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev. 17, Habas, R., Kato, Y., and He, X. (2001). Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107, Tahinci, E., and Symes, K. (2003). Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation. Dev. Biol. 259, Choi, S.C., and Han, J.K. (2002). Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway. Dev. Biol. 244, Marsden, M., and DeSimone, D.W. (2001). Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin. Development 128, Marsden, M., and DeSimone, D.W. (2003). Integrin-ECM interactions regulate cadherin-dependent cell adhesion and are required for convergent extension in Xenopus. Curr. Biol. 13, Zhong, C., Chrzanowska-Wodnicka, M., Brown, J., Shaub, A., Belkin, A.M., and Burridge, K. (1998). Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J. Cell Biol. 141, Davidson, L.A., Keller, R., and DeSimone, D.W. (2004). Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis. Dev. Dyn. 231, Wheeler, G.N., and Hoppler, S. (1999). Two novel Xenopus frizzled genes expressed in developing heart and brain. Mech. Dev. 86,