Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation

Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin protein Daam1 and an effector molecule XProfilin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProfilin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProfilin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1 regulates blastopore closure, overexpression or depletion of XProfilin2 specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1. Together, our studies demonstrate that XProfilin2 and XProfilin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.     

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.     

Daam2 is required for dorsal patterning via modulation of canonical Wnt signaling in the developing spinal cord

The Daam family of proteins consists of Daam1 and Daam2. Although Daam1 participates in noncanonical Wnt signaling during gastrulation, Daam2 function remains completely uncharacterized. Here we describe the role of Daam2 in canonical Wnt signal transduction during spinal cord development. Loss-of-function studies revealed that Daam2 is required for dorsal progenitor identities and canonical Wnt signaling. These phenotypes are rescued by β-catenin, demonstrating that Daam2 functions in dorsal patterning through the canonical Wnt pathway. Complementary gain-of-function studies demonstrate that Daam2 amplifies Wnt signaling by potentiating ligand activation. Biochemical examination found that Daam2 association with Dvl3 is required for Wnt activity and dorsal patterning. Moreover, Daam2 stabilizes Dvl3/Axin2 binding, resulting in enhanced intracellular assembly of Dvl3/Axin2 complexes. These studies demonstrate that Daam2 modulates the formation of Wnt receptor complexes, revealing new insight into the functional diversity of Daam proteins and how canonical Wnt signaling contributes to pattern formation in the developing spinal cord.     

Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1.

Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. Here we report that in human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1. Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of beta-catenin signaling. Our study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction.     

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis

Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.     

Identification and comparative expression analyses of Daam genes in mouse and Xenopus

During vertebrate embryogenesis, secreted Wnt molecules regulate cell fates by signaling through the canonical pathway mediated by beta-catenin, and regulate planar cell polarity (PCP) and convergent extension movements through alternative pathways. The phosphoprotein Dishevelled (Dsh/Dvl) is a Wnt signal transducer thought to function in all Wnt signaling pathways. A recently identified member of the Formin family, Daam (Dishevelled--associated activator of morphogenesis), regulates the morphogenetic movements of vertebrate gastrulation in a Wnt-dependent manner through direct interactions with Dsh/Dvl and RhoA. We describe two mouse Daam cDNAs, mDaam1 and mDaam2, which encode proteins characterized by highly conserved formin homology domains and which are expressed in complementary patterns during mouse development. Cross-species comparisons indicate that the expression domains of Xenopus Daam1 (XDaam1) mirror mDaam1 expression. Our results demonstrate that Daams are expressed in tissues known to require Wnts and are consistent with Daams being effectors of Wnt signaling during vertebrate development.     

Role of PKA as a negative regulator of PCP signaling pathway during Xenopus gastrulation movements

Convergent extension (CE) movements in gastrulation are essential for the establishment of the body axis during early vertebrate development. Although the precise molecular mechanisms of CE movements are not clearly understood, noncanonical Wnt pathway is known to be important for the control of CE movements. Here, we present evidence that PKA is implicated in noncanonical Wnt pathway. Overexpression and specific depletion of PKA inhibit CE movements. PKA depletion also disrupts cell morphology, protrusive activity, and cortical actin formation in dorsal mesodermal cells. Moreover, PKA activity is negatively regulated by major components of planar cell polarity (PCP) pathway. In line with this, overexpression of PKA can rescue the inhibition of CE movements caused by overexpression of these molecules. We also demonstrate that this regulation of PKA activity is dependent upon Galphai signaling. As a negative component of PCP signaling, PKA inhibits not only the activation of RhoA and JNK but also the Dsh-Daam1-RhoA complex formation which is essential for the regulation of RhoA activity. Together, our study suggests a molecular pathway from Wnt/Dsh/PKA signaling to Rho activation in PCP signaling.     

Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1

The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1.     

Regulation of convergence and extension movements during vertebrate gastrulation by the Wnt/PCP pathway

Vertebrate gastrulation entails massive cell movements that establish and shape the germ layers. During gastrulation, the individual cell behaviors are strictly coordinated in time and space by various signaling pathways. These pathways instruct the cells about proliferation, shape, fate and migration into proper location. Convergence and extension (C&E) movements during vertebrate gastrulation play a major role in the shaping of the embryonic body. In vertebrates, the Wnt/Planar Cell Polarity (Wnt/PCP) pathway is a key regulator of C&E movements, essential for several polarized cell behaviors, including directed cell migration, and mediolateral and radial cell intercalation. However, the molecular mechanisms underlying the acquisition of Planar Cell Polarity by highly dynamic mesenchymal cells engaged in C&E are still not well understood. Here we review new evidence implicating the Wnt/PCP pathway in specific cell behaviors required for C&E during zebrafish gastrulation, in comparison to other vertebrates. We also discuss findings on the molecular regulation and the interaction of the Wnt/PCP pathway with other signaling pathways during gastrulation movements.     

The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization

Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.     

Essential roles of G{alpha}12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements

Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.     

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 Ca²⁺- and Mg²⁺-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg²⁺ supplementation or by expression of the Mg²⁺ transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg²⁺ in Rac-dependent polarized cell movements during vertebrate gastrulation.     

Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin

Wnt11 plays a central role in tissue morphogenesis during vertebrate gastrulation, but the molecular and cellular mechanisms by which Wnt11 exerts its effects remain poorly understood. Here, we show that Wnt11 functions during zebrafish gastrulation by regulating the cohesion of mesodermal and endodermal (mesendodermal) progenitor cells. Importantly, we demonstrate that Wnt11 activity in this process is mediated by the GTPase Rab5, a key regulator of early endocytosis, as blocking Rab5c activity in wild-type embryos phenocopies slb/wnt11 mutants, and enhancing Rab5c activity in slb/wnt11 mutant embryos rescues the mutant phenotype. In addition, we find that Wnt11 and Rab5c control the endocytosis of E-cadherin and are required in mesendodermal cells for E-cadherin-mediated cell cohesion. Together, our results suggest that Wnt11 controls tissue morphogenesis by modulating E-cadherin-mediated cell cohesion through Rab5c, a novel mechanism of Wnt signaling in gastrulation.     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

Def6 is required for convergent extension movements during zebrafish gastrulation downstream of Wnt5b signaling

During gastrulation, convergent extension (CE) cell movements are regulated through the non-canonical Wnt signaling pathway. Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement. Rho GTPases are bi-molecular switches that are inactive in their GDP-bound stage but can be activated to bind GTP through guanine nucleotide exchange factors (GEFs). Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation. Def6 morphants exhibit broadened and shortened body axis with normal cell fate specification, reminiscent of the zebrafish mutants silberblick and pipetail that lack Wnt11 or Wnt5b, respectively. Indeed, def6 morphants phenocopy Wnt5b mutants and ectopic overexpression of def6 essentially rescues Wnt5b morphants, indicating a novel role for def6 as a central GEF downstream of Wnt5b signaling. In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.     

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.     

Wnt signalling regulates paxillin ubiquitination essential for mesodermal cell motility

Gastrulation movements are critical for establishing the three germ layers and the architecture of vertebrate embryos. During Xenopus laevis gastrulation, mesodermal tissue migrates on the blastocoel roof and elongates along the antero-posterior axis. During this process, cells in the dorsal mesoderm are polarized and intercalate with each other, which is defined as convergent extension and is known to be regulated by the non-canonical Wnt pathway. Here, we show that paxillin plays an essential role in this process. Paxillin is a focal-adhesion associated protein implicated in the regulation of actin cytoskeletal organization and cell motility, but its role in Xenopus embryogenesis has not yet been clarified. We demonstrate that the Wnt pathway controls the ubiquitination and stability of paxillin, and that this regulatory mechanism is essential for convergent extension movements. We identified a RING finger protein XRNF185, which physically binds to paxillin and the proteasome. XRNF185 destabilizes paxillin at focal adhesions and promotes mesodermal cell migration during convergent extension. We propose a mechanism to regulate gastrulation movements that involves paxillin ubiquitination and stability controlled by Wnt signalling.     

The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.     

Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm

Formation of mesoderm from the pluripotent epiblast depends upon canonical Wnt/beta-catenin signaling, although a precise molecular basis for this requirement has not been established. To develop a robust model of this developmental transition, we examined the role of Wnt signaling during the analogous stage of embryonic stem cell differentiation. We show that the kinetics of Wnt ligand expression and pathway activity in vitro mirror those found in vivo. Furthermore, inhibition of this endogenous Wnt signaling abrogates the functional competence of differentiating ES cells, reflected by their failure to generate Flk1(+) mesodermal precursors and subsequent mature mesodermal lineages. Microarray analysis at various times during early differentiation reveal that mesoderm- and endoderm-associated genes fail to be induced in the absence of Wnt signaling, indicating a lack of germ layer induction that normally occurs during gastrulation in vivo. The earliest genes displaying Wnt-dependent expression, however, were those expressed in vivo in the primitive streak. Using an inducible form of stabilized beta-catenin, we find that Wnt activity, although required, does not autonomously promote primitive streak-associated gene expression in vitro. Our results suggest that Wnt signaling functions in this model system to regulate the thresholds or stability of responses to other effector pathways and demonstrate that differentiating ES cells represent a useful model system for defining complex regulatory interactions underlying primary germ layer induction.