The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

BACKGROUND: The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. RESULTS: Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. CONCLUSIONS: Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Formin Binding Protein 1 (FNBP1) regulates non-canonical Wnt signaling and vertebrate gastrulation (12 words).

The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1 and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.

Collagen Nanoyarns: Hierarchical Three-Dimensional Biomaterial Constructs.

Hierarchical fibrous scaffolds (HFS) consist of nanoscale fibers arranged in larger macroscale structures, much in the same pattern as in native tissue such as tendon and bone. Creation of continuous macroscale nanofiber yarns has been made possible using modified electrospinning set-ups that combine electrospinning with techniques such as twisting, drawing, and winding. In this paper, a modified electrospinning setup was used to create continuous yarns of twisted type I collagen nanofibers, also known as collagen nanoyarns (CNY), from collagen solution prepared in acetic acid. Fabricated CNYs were cross-linked and characterized using SEM imaging and mechanical testing, while denaturation of collagen and dissolution of the scaffolds were assessed using circular dichroism (CD) and UV-vis spectroscopy, respectively. HeLa cells were then cultured on the nanoyarns for 24 h to assess cell adhesion on the scaffolds. Scanning electron micrographs revealed a twisted nanofiber morphology with an average nanofiber diameter of 213 ± 60 nm and a yarn diameter of 372 ± 23 µm that shrank by 35% after covalent cross-linking. Structural denaturation assessment of native collagen using circular dichroism (CD) spectroscopy showed that 60% of the triple-helical collagen content in CNYs was retained. Cross-linking of CNYs significantly improved their mechanical properties as well as stability in buffered saline with no sign of degradation for 14 days. In addition, CNY strength and stiffness increased significantly with cross-linking although in the wet state, significant loss in these properties, with a corresponding increase in elasticity, was observed. HeLa cells cultured on cross-linked CNYs for 24 h adhered to the yarn surface and oriented along the nanofiber alignment axis, displaying the characteristic spindle-like morphology of cells grown on surfaces with aligned topography. Collectively, the results demonstrate the promising potential of collagen nanoyarns as a new class of shapable biomaterial scaffold and building block for generating macroscale fiber-based tissues.

Biochemical Assays to Detect Activation of Small GTPases Rho, Rac, and Cdc42 during Morphogenesis.

Wnt/Frizzled (Fz) signaling controls developmental, physiological, and pathological processes through several distinct pathways. Wnt/Fz activation of the small GTPases Rho, Rac, and Cdc42, is one key mechanism that regulates cell polarity and migration during vertebrate gastrulation. In this chapter, we describe biochemical assays for detection of Wnt/Fz-mediated activation of Rho, Rac and Cdc42 in both mammalian cells and Xenopus embryo explants.

Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes.

Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

Placental defects lead to embryonic lethality in mice lacking the Formin and PCP proteins Daam1 and Daam2.

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.

A Nonredundant Role for the TRPM6 Channel in Neural Tube Closure.

In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6's close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.

Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis.

Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl â†’ Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.

Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis.

Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression.

DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly.

Wnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development.

Osteoblast biocompatibility of premineralized, hexamethylene-1,6-diaminocarboxysulfonate crosslinked chitosan fibers.

Biopolymer-ceramic composites are thought to be particularly promising materials for bone tissue engineering as they more closely mimic natural bone. Here, we demonstrate the fabrication by electrospinning of fibrous chitosan-hydroxyapatite composite scaffolds with low (1 wt %) and high (10 wt %) mineral contents. Scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS) and unidirectional tensile testing were performed to determine fiber surface morphology, elemental composition, and tensile Young's modulus (E) and ultimate tensile strength (σUTS ), respectively. EDS scans of the scaffolds indicated that the fibers, crosslinked with either hexamethylene-1,6-diaminocarboxysulfonate (HDACS) or genipin, have a crystalline hydroxyapatite mineral content at 10 wt % additive. Moreover, FESEM micrographs showed that all electrospun fibers have diameters (122-249 nm), which fall within the range of those of fibrous collagen found in the extracellular matrix of bone. Young's modulus and ultimate tensile strength of the various crosslinked composite compositions were in the range of 116-329 MPa and 2-15 MPa, respectively. Osteocytes seeded onto the mineralized fibers were able to demonstrate good biocompatibility enhancing the potential use for this material in future bone tissue engineering applications.

Osteoblast biocompatibility of novel chitosan crosslinker, hexamethylene-1,6-diaminocarboxysulfonate.

Chitosan is a naturally occurring polysaccharide, which has proven to be an attractive candidate for bone tissue engineering, due to its ability to promote osteoblast mineralization. Electrospinning has become a well-established cell scaffold processing technique, as it produces a high surface area to volume fibrous material that can mimic the three dimensionality of the extracellular matrix of a cell. In this study, we have investigated the osteoblast response to two different chemically crosslinked (hexamethylene-1,6-diaminocarboxysulfonate (HDACS) and genipin) electrospun chitosan scaffolds and their film counterparts in order to determine how material chemistry influences cellular behavior in conjunction with material topology. In addition, material properties of each fiber scaffold such as porosity and tensile strength were considered. MLO-A5 osteoblast cells grown on chitosan-HDACS scaffolds were found to display a more organized cellular network, along with significantly more filopodia extensions, compared to those grown on chitosan-genipin scaffolds. After 2 days of growth on chitosan-HDACS fibers, a higher level of alkaline phosphatase expression in MLO-A5 cells was reported compared to that of either chitosan-genipin fibers or films. These results indicate that not only chemistry, but also surface topology is an important effecter of cellular behavior. Ultimately, chitosan-HDACS fiber scaffolds provided an adequate substrate for osteoblast attachment and proliferation.

The increase in maternal expression of axin1 and axin2 contribute to the zebrafish mutant ichabod ventralized phenotype.

ß-Catenin is a central effector of the Wnt pathway and one of the players in Ca(+)-dependent cell-cell adhesion. While many wnts are present and expressed in vertebrates, only one ß-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for ß-catenin. The maternal recessive mutation ichabod presents very low levels of ß-catenin2 that in turn affects dorsal axis formation, suggesting that ß-catenin1 is incapable to compensate for ß-catenin2 loss and raising the question of whether these two ß-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for ß-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that ß-catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of ß-catenin regulatory pathway, including ß-catenin1, are more abundant than in the Wt embryo. Increased levels of ß-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that ß-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3ß-independent mechanism that required Axin's RGS domain function.

Custos controls ß-catenin to regulate head development during vertebrate embryogenesis.

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of ß-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited ß-catenin/TCF-dependent transcription. Custos binds to ß-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of ß-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-ß-catenin signal transduction cascade.

Magnesium and embryonic development.

Important for energy metabolism, neurotransmission, bone stability, and other cellular functions, Mg(2+) has well-established and undisputedly critical roles in adult tissues. Its contributions to early embryonic development are less clearly understood. For decades it has been known that gestational Mg(2+) deficiency in rodents produces teratogenic effects. More recent studies have linked deficiency in this vital cation to birth defects in humans, including spina bifida, a neural fold closure defect in humans that occurs at an average rate of 1 per 1000 pregnancies. The first suggestion that Mg(2+) may be playing a more specific role in early development arose from studies of the TRPM7 and TRPM6 ion channels. TRPM7 and TRPM6 are divalent-selective ion channels in possession of their own kinase domains that have been implicated in the control of Mg(2+) homeostasis in vertebrates. Disruption of the functions of these ion channels in mice as well as in frogs interferes with gastrulation, a pivotal process during early embryonic development that executes the emergence of the body plan and closure of the neural tube. Surprisingly, gastrulation defects produced by depletion of TRPM7 can be prevented by Mg(2+) supplementation, indicating an essential role for Mg(2+) in gastrulation and neural fold closure. The aim of this review is to summarize the data emerging from molecular genetic, biochemical and electrophysiological studies of TRPM6 and TRPM7 and provide a model of how Mg(2+), through these unique channel-kinases, may be impacting early embryonic development.

Activation and function of small GTPases Rho, Rac, and Cdc42 during gastrulation.

Gastrulation is comprised of a series of cell polarization and directional cell migration events that establish the physical body plan of the embryo. One of the major ligand-based pathways that has emerged to play crucial roles in the regulation of gastrulation is the non-canonical Wnt signaling pathway. This aspect of Wnt signaling is comprised of a number of signaling branches that are subsequently integrated for the regulation of changes to the actin cytoskeleton during cell polarization and cell migration during vertebrate gastrulation. The Rho family of small GTPases are activated and required for non-canonical Wnt signaling during gastrulation, and in this chapter, we describe biochemical assays for the detection of Wnt-mediated activation of Rho, Rac, and Cdc42 in both mammalian cells and Xenopus embryo explants.

Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.

Canonical ß-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

MIM regulates vertebrate neural tube closure.

Neural tube closure is a critical morphogenetic event that is regulated by dynamic changes in cell shape and behavior. Although previous studies have uncovered a central role for the non-canonical Wnt signaling pathway in neural tube closure, the underlying mechanism remains poorly resolved. Here, we show that the missing in metastasis (MIM; Mtss1) protein, previously identified as a Hedgehog response gene and actin and membrane remodeling protein, specifically binds to Daam1 and couples non-canonical Wnt signaling to neural tube closure. MIM binds to a conserved domain within Daam1, and this interaction is positively regulated by Wnt stimulation. Spatial expression of MIM is enriched in the anterior neural plate and neural folds, and depletion of MIM specifically inhibits anterior neural fold closure without affecting convergent extension movements or mesoderm cell fate specification. Particularly, we find that MIM is required for neural fold elevation and apical constriction along with cell polarization and elongation in both the superficial and deep layers of the anterior neural plate. The function of MIM during neural tube closure requires both its membrane-remodeling domain and its actin-binding domain. Finally, we show that the effect of MIM on neural tube closure is not due to modulation of Hedgehog signaling in the Xenopus embryo. Together, our studies define a morphogenetic pathway involving Daam1 and MIM that transduces non-canonical Wnt signaling for the cytoskeletal changes and membrane dynamics required for vertebrate neural tube closure.

TRPM7 regulates polarized cell movements.

TRPM7 (transient receptor potential melastatin 7) is a Ca²+- and Mg²+-permeant ion channel in possession of its own kinase domain. As a kinase, the protein has been linked to the control of actomyosin contractility, whereas the channel has been found to regulate cell adhesion as well as cellular Mg²+ homoeostasis. In the present study we show that depletion of TRPM7 by RNA interference in fibroblasts alters cell morphology, the cytoskeleton, and the ability of cells to form lamellipodia and to execute polarized cell movements. A pulldown-purification assay revealed that knockdown of TRPM7 prevents cells from activating Rac and Cdc42 (cell division cycle 42) when stimulated to migrate into a cellular wound. Re-expression of TRPM7 reverses these phenotypic changes, as does, unexpectedly, expression of a kinase-inactive mutant of TRPM7. Surprisingly, expression of the Mg²+ transporter SLC41A2 (solute carrier family 41 member 2) is also effective in restoring the change in cell morphology, disruption of the cytoskeleton and directional cell motility caused by depletion of the channel-kinase. The results of the present study uncover an essential role for Mg²+ in the control of TRPM7 over the cytoskeleton and its ability to regulate polarized cell movements.

TRPM7 regulates gastrulation during vertebrate embryogenesis.

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca(2+)- and Mg(2+)-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg(2+) supplementation or by expression of the Mg(2+) transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg(2+) in Rac-dependent polarized cell movements during vertebrate gastrulation.