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.     

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell-cell and cell-matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.     

Mesoderm-independent regulation of gastrulation movements by the Src tyrosine kinase in Xenopus embryo

In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

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.     

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.     

Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus

We compared the type and patterning of morphogenic cell behaviors driving convergent extension of the Xenopus neural plate in the presence and absence of persistent vertical signals from the mesoderm by videorecording explants of deep neural tissue with involuted mesoderm attached and of deep neural tissue alone. In deep neural-over-mesoderm explants, neural plate cells express monopolar medially directed motility and notoplate cells express randomly oriented motility, two new morphogenic cell behaviors. In contrast, in deep neural explants (without notoplate), all cells express bipolar mediolateral cell motility. Deep neural-over-mesoderm and deep neural explants also differ in degree of neighbor exchange during mediolateral cell intercalation. In deep neural-over-mesoderm explants, cells intercalate conservatively, whereas in deep neural explants cells intercalate more promiscuously. Last, in both deep neural-over-mesoderm and deep neural explants, morphogenic cell behaviors differentiate in an anterior-to-posterior and lateral-to-medial progression. However, in deep neural-over-mesoderm explants, morphogenic behaviors first differentiate in intervals along the anteroposterior axis, whereas in deep neural explants, morphogenic behaviors differentiate continuously from the anterior end of the tissue posteriorly. These results describe new morphogenic cell behaviors driving neural convergent extension and also define roles for signals from the mesoderm, up to and beyond late gastrulation, in patterning these cell behaviors.     

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.     

Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis

During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.     

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.     

Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants

We make use of a novel system of explant culture and high resolution video-film recording to analyse for the first time the cell behaviour underlying convergent extension and segmentation in the somitic mesoderm of Xenopus. We find that a sequence of activities sweeps through the somitic mesoderm from anterior to posterior during gastrulation and neurulation, beginning with radial cell intercalation or thinning, continuing with mediolateral intercalation and cell elongation, and culminating in segmentation and somite rotation. Radial intercalation at the posterior tip lengthens the tissue, while mediolateral intercalation farther anterior converges it toward the midline. This extension of the somitic mesoderm helps to elongate the dorsal side of intact neurulae. By separating tissues, we demonstrate that cell rearrangement is independent of the notochord, but radial intercalation - and thus the bulk of extension - requires the presence of an epithelium, either endodermal or ectodermal. Segmentation, on the other hand, can proceed in somitic mesoderm isolated at the end of gastrulation. Finally, we discuss the relationship between cell rearrangement and segmentation.     

PDGF-A controls mesoderm cell orientation and radial intercalation during Xenopus gastrulation

Radial intercalation is a common, yet poorly understood, morphogenetic process in the developing embryo. By analyzing cell rearrangement in the prechordal mesoderm during Xenopus gastrulation, we have identified a mechanism for radial intercalation. It involves cell orientation in response to a long-range signal mediated by platelet-derived growth factor (PDGF-A) and directional intercellular migration. When PDGF-A signaling is inhibited, prechordal mesoderm cells fail to orient towards the ectoderm, the endogenous source of PDGF-A, and no longer migrate towards it. Consequently, the prechordal mesoderm fails to spread during gastrulation. Orientation and directional migration can be rescued specifically by the expression of a short splicing isoform of PDGF-A, but not by a long matrix-binding isoform, consistent with a requirement for long-range signaling.     

Epithelial Cell Wedging and Neural Trough Formation Are Induced Planarly inXenopus,without Persistent Vertical Interactions with Mesoderm

In this study we investigate the induction of the cell behaviors underlying neurulation in the frog,Xenopus laevis.Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Kelleret al.,1992,Dev. Dyn.193, 218–234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a “giant sandwich” explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.     

Signal transduction via platelet-derived growth factor receptors

Platelet-derived growth factor (PDGF) exerts its stimulatory effects on cell growth and motility by binding to two related protein tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation, allowing binding and activation of cytoplasmic SH2-domain containing signal transduction molecules. Thereby, a number of different signaling pathways are initiated leading to cell growth, actin reorganization migration and differentiation. Recent observations suggest that extensive cross-talk occurs between different signaling pathways, and that stimulatory signals are modulated by inhibitory signals arising in parallel.     

Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulation

Gastrulation is a dynamic tissue-remodeling process occurring during early development and fundamental to the later organogenesis. It involves both chemical signals and physical factors. Although much is known about the molecular pathways involved, the roles of physical forces in regulating cellular behavior and tissue remodeling during gastrulation have just begun to be explored. Here, we characterized the force generated by the leading edge mesoderm (LEM) that migrates preceding axial mesoderm (AM), and investigated the contribution of LEM during Xenopus gastrulation. First, we constructed an assay system using micro-needle which could measure physical forces generated by the anterior migration of LEM, and estimated the absolute magnitude of the force to be 20–80 nN. Second, laser ablation experiments showed that LEM could affect the force distribution in the AM (i.e. LEM adds stretch force on axial mesoderm along anterior–posterior axis). Third, migrating LEM was found to be necessary for the proper gastrulation cell movements and the establishment of organized notochord structure; a reduction of LEM migratory activity resulted in the disruption of mediolateral cell orientation and convergence in AM. Finally, we found that LEM migration cooperates with Wnt/PCP to form proper notochord.     

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.     

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.