PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.     

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.     

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.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Convergence and extension movements mediate the specification and fate maintenance of zebrafish slow muscle precursors

During vertebrate gastrulation, concurrent inductive events and cell movements fashion the body plan. Convergence and extension (C&E) gastrulation movements narrow the vertebrate embryonic body mediolaterally while elongating it rostrocaudally. Segmented somites are shaped and positioned by C&E alongside the notochord and differentiate into skeleton, fast, and slow muscles during somitogenesis. In zebrafish, simultaneous inactivation of non-canonical Wnt signaling components Knypek and Trilobite strongly impairs C&E gastrulation movements. Here we show that knypek;trilobite double mutants exhibit a severe deficit in slow muscles and their precursor, adaxial cells, revealing essential roles of C&E movements in adaxial cell development. Adaxial cells become distinguishable in the presomitic mesoderm during late gastrulation by their expression of myogenic factors and axial-adjacent position. Using cell tracing analyses and genetic manipulations, we demonstrate that C&E movements regulate the number of prospective adaxial cells specified during gastrulation by determining the size of the interface between the inductive axial and target presomitic tissues. During segmentation, when the range of Hedgehog signaling from the axial tissue declines, tight apposition of prospective adaxial cells to the notochord, which is achieved by convergence movements, is necessary for their continuous Hedgehog reception and fate maintenance. We provide direct evidence to show that the deficiency of adaxial cells in knypek;trilobite double mutants is due to impaired C&E movements, rather than an alteration in Hedgehog signal and its reception, or a cell-autonomous requirement for Knypek and Trilobite in adaxial cell development. Our results underscore the significance of precise coordination between cell movements and inductive tissue interactions during cell fate specification.     

Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation

During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas Dishevelled is enriched posteriorly. Asymmetrical localization of Prickle and Dishevelled to the opposite cell edges in zebrafish gastrula parallels their distribution in fly, and suggests that noncanonical Wnt signaling defines distinct anterior and posterior cell properties to bias cell intercalations.     

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.     

Phosphoinositide 3-kinase is required for process outgrowth and cell polarization of gastrulating mesendodermal cells

BACKGROUND: During vertebrate gastrulation, cell polarization and migration are core components in the cellular rearrangements that lead to the formation of the three germ layers, ectoderm, mesoderm, and endoderm. Previous studies have implicated the Wnt/planar cell polarity (PCP) signaling pathway in controlling cell morphology and movement during gastrulation. However, cell polarization and directed cell migration are reduced but not completely abolished in the absence of Wnt/PCP signals; this observation indicates that other signaling pathways must be involved. RESULTS: We show that Phosphoinositide 3-Kinases (PI3Ks) are required at the onset of zebrafish gastrulation in mesendodermal cells for process formation and cell polarization. Platelet Derived Growth Factor (PDGF) functions upstream of PI3K, while Protein Kinase B (PKB), a downstream effector of PI3K activity, localizes to the leading edge of migrating mesendodermal cells. In the absence of PI3K activity, PKB localization and cell polarization are strongly reduced in mesendodermal cells and are followed by slower but still highly coordinated and directed movements of these cells. CONCLUSIONS: We have identified a novel role of a signaling pathway comprised of PDGF, PI3K, and PKB in the control of morphogenetic cell movements during gastrulation. Furthermore, our findings provide insight into the relationship between cell polarization and directed cell migration at the onset of zebrafish gastrulation.     

FGF signal interpretation is directed by Sprouty and Spred proteins during mesoderm formation

Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.     

Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation

The zyxin-related LPP protein is localized at focal adhesions and cell–cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior–posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.     

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.     

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.     

Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity

Little is known about how protocadherins function in cell adhesion and tissue development. Paraxial protocadherin (PAPC) controls cell sorting and morphogenetic movements in the Xenopus laevis embryo. We find that PAPC mediates these functions by down-regulating the adhesion activity of C-cadherin. Expression of exogenous C-cadherin reverses PAPC-induced cell sorting and gastrulation defects. Moreover, loss of endogenous PAPC results in elevated C-cadherin adhesion activity in the dorsal mesoderm and interferes with the normal blastopore closure, a defect that can be rescued by a dominant-negative C-cadherin mutant. Importantly, activin induces PAPC expression, and PAPC is required for activin-induced regulation of C-cadherin adhesion activity and explant morphogenesis. Signaling through Frizzled-7 is not required for PAPC regulation of C-cadherin, suggesting that C-cadherin regulation and Frizzled-7 signaling are two distinct branches of the PAPC pathway that induce morphogenetic movements. Thus, spatial regulation of classical cadherin adhesive function by local expression of a protocadherin is a novel mechanism for controlling cell sorting and tissue morphogenesis.     

Gastrulation in Xenopus laevis: involution—a current view

In this article, we describe some of the morphogenetic movements reshaping the Xenopus laevis embryo during gastrulation. We have learned a great deal about these movements in recent years through advances made in explant culture techniques. Here, we will focus on involution, the process by which mesoderm is internalized and placed in between ectoderm and endoderm. Our aim is to present our current view of how involution takes place in the dorsal involuting marginal zone of the Xenopus embryos.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

ANR5, an FGF target gene product, regulates gastrulation in Xenopus

Gastrulation is a morphogenetic process in which tightly coordinated cell and tissue movements establish the three germ layers (ectoderm, mesoderm, and endoderm) to define the anterior-to-posterior embryonic organization [1]. To elicit this movement, cells modulate membrane protrusions and undergo dynamic cell interactions. Here we report that ankyrin repeats domain protein 5 (xANR5), a novel FGF target gene product, regulates cell-protrusion formation and tissue separation, a process that develops the boundary between the ectoderm and mesoderm [2, 3], during Xenopus gastrulation. Loss of xANR5 function by antisense morpholino oligonucleotide (MO) caused a short trunk and spina bifida without affecting mesodermal gene expressions. xANR5-MO also blocked elongation of activin-treated animal caps (ACs) and tissue separation. The dorsal cells of xANR5-MO-injected embryos exhibited markedly reduced membrane protrusions, which could be restored by coinjecting active Rho. Active Rho also rescued the xANR5-MO-inhibited tissue separation. We further demonstrated that xANR5 interacted physically and functionally with paraxial protocadherin (PAPC), which has known functions in cell-sorting behavior, tissue separation, and gastrulation cell movements [4-6], to regulate early morphogenesis. Our findings reveal for the first time that xANR5 acts through Rho to regulate gastrulation and is an important cytoplasmic partner of PAPC, whose cytoplasmic partner was previously unknown.