Xenopus Gastrulation Research Articles

The involvement of Eph–Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph–ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

P05. Mechanical force generated by leading edge mesoderm modulates collective cell polarization in axial mesoderm during Xenopus gastrulation

Gastrulation is one of the most important processes during the morphogenesis of early embryo, involving dynamic cell shape change and migration. In chordates, notochord, a rod-shape structure that contributes to body extension and neural tube formation, is formed by collective cell movements of the axial mesoderm (AM) through gastrulation. During notochord formation, AM cells become narrow along mediolateral (M–L) axis and such polarized cells intercalate each other to extend the tissue along anterior–posterior (A–P) axis. This process, called convergent extension (CE), requires the establishment of cell polarity at the same time and in the same direction in AM. Although planar cell polarity (PCP) pathway is essential for cell polarization and regulation of CE, little is known about how the polarity of AM cells is established collectively at the beginning of CE. During Xenopus gastrulation, leading edge mesoderm (LEM), positioned in front of the AM, attach to the blastocoel roof and migrate towards the animal pole. The LEM contributes to the directional migration of involuting marginal zone cells. Therefore, we hypothesized that anterior migration of the LEM generate the pulling force along A–P direction, and the generated force stretch AM cells to establish M–L cell polarity. To test this hypothesis, we first performed mesoderm migration assay in vitro and found that the LEM migrated faster than the AM. We measured the force generated by the LEM in vitro using thin glass pipette. The estimated force varied but ranged from tens to hundreds of nano-newtons. These results suggest that the LEM generate the pulling force to the AM in a significant order of force magnitude. Next, to test whether the pulling force induces collective cell polarization in the AM, we developed stretch assay system using a silicone chamber. To determine the magnitude of stretch force applied to the activin-induced AM on the scale same as we observed, we measured the Young’s modulus of the AM using glass pipette and calculated the force by formula of Hooke’s law. Using these methods, we estimated that 5–15% stretch is probably equal to LEM-generated force. We are currently examining the effect of such stretch on AM with comparable magnitudes of force generated by LEM, and determine if the stretching affects polarity formation process. This study might contribute to better understanding of the distribution of force fields and the function of mechanical factors in animal development.

The cytoplasmic tyrosine kinase Arg regulates Xenopus gastrulation via the adaptor protein CrkII

The cytoplasmic tyrosine kinase Arg regulates Xenopus gastrulation via the adaptor protein CrkII

Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis.

Xclaudin 1 is required for the proper gastrulation in Xenopus laevis

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.

XGit2 and xRhoGAP 11A regulate convergent extension and tissue separation in Xenopus gastrulation

In a microarray-based screen for genes that are involved in tissue separation downstream of Paraxial Protocadherin (PAPC) and Frizzled-7 (Fz7)-mediated signaling we identified xGit2 and xRhoGAP 11A, two GTPase-activating proteins (GAP) for small GTPases. xGit2 and xRhoGAP 11A are expressed in the dorsal ectoderm, and their transcription is downregulated in the involuting dorsal mesoderm by PAPC and Fz7. Overexpression of xGit2 and xRhoGAP 11A inhibits Rho activity and impairs convergent extension movements as well as tissue separation behaviour. We propose that Rho activity in the involuting mesoderm is enhanced through inhibition of xGit2 and xRhoGAP 11A transcription by PAPC and Fz7. By this mechanism xRhoGAP 11A and xGit2 are restricted to the dorsal ectoderm, while Rho signaling is inhibited.

Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation.

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates mechanical forces through cell-cell contact that initiates coordinated cell polarization through a transient increase in intracellular calcium.

Endocytosis Is Required for Efficient Apical Constriction during Xenopus Gastrulation

Coordinated apical constriction (AC) in epithelial sheets drives tissue invagination [1, 2] and is required for diverse morphogenetic movements such as gastrulation [3], neurulation [4, 5], and organogenesis [6]. We showed previously that actomyosin contractility drives AC in Xenopus laevis bottle cells [7]; however, it remained unclear whether it does so in concert with other processes. Here we report that endocytosis-driven membrane remodeling is required for efficient AC. We found endosomes exclusively in bottle cells in the early gastrula. Disrupting endocytosis with dominant-negative dynamin or rab5 perturbed AC, with a significant decrease in constriction rate late in the process, suggesting that endocytosis operates downstream of actomyosin contractility to remove excess membrane. Additionally, disrupting endocytosis during neurulation inhibits AC in hingepoint cells, resulting in neural tube closure defects. Thus, membrane remodeling during AC could be a general mechanism to achieve efficient invagination in embryos.

PDGF-A interactions with fibronectin reveal a critical role for heparan sulfate in directed cell migration during Xenopus gastrulation.

Platelet-derived growth factor (PDGF) signaling is essential for processes involving cell motility and differentiation during embryonic development in a wide variety of organisms including the mouse, frog, zebrafish, and sea urchin. In early Xenopus laevis embryos, PDGF-AA provides guidance cues for the migration of anterior mesendoderm cells as they move across a fibronectin-rich extracellular matrix. The long form of PDGF-A includes a positively charged carboxyl-terminal retention motif that can interact with the extracellular matrix and heparan sulfate proteoglycans (HSPGs). In this study we demonstrate that PDGF-AA binds directly to fibronectin and that this association is greatly enhanced by heparin. The PDGF-AA-fibronectin binding occurs across a broad range of pHs (5.5-9), which is significant because the PDGF-guided migration of Xenopus mesendoderm cells occurs under basic extracellular conditions (pH 8.4). We further demonstrate that endogenous HSPG's are required for the PDGF-AA-guided mesendoderm movement, suggesting an in vivo role for HSPGs in mediating the interaction between PDGF-AA and fibronectin.

Highlights in DD

Highlights in <i>DD</i>

Cullin Mediates Degradation of RhoA through Evolutionarily Conserved BTB Adaptors to Control Actin Cytoskeleton Structure and Cell Movement

Cul3, a Cullin family scaffold protein, is thought to mediate the assembly of a large number of SCF (Skp1-Cullin1-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Cul3 controls early embryonic development in several genetic models through mechanisms not understood. Very few functional substrate/adaptor pairs for Cul3 ubiquitin ligases have been identified. Here, we show that Cul3 knockdown in human cells results in abnormal actin stress fibers and distorted cell morphology, owing to impaired ubiquitination and degradation of small GTPase RhoA. We identify a family of RhoA-binding BTB domain adaptors conserved from insects to mammals, designated BACURDs. BACURDs form ubiquitin ligase complexes, which selectively ubiquitinate RhoA, with Cul3. Dysfunction of the Cul3/BACURD complex decreases cell migration potential and impairs RhoA-mediated convergent extension movements during Xenopus gastrulation. Our studies reveal a previously unknown mechanism for controlling RhoA degradation and regulating RhoA function in various biological contexts, which involves a Cul3/BACURD ubiquitin ligase complex.

Imaging morphogenesis, in Xenopus with Quantum Dot nanocrystals

Mesoderm migration is a well studied morphogenetic movement that takes place during Xenopus gastrulation. The study of mesoderm migration and other morphogenetic movements has been primarily based on in vitro assays due to the inability to image deep tissue movements in the opaque embryo. We are the first to report the use of Near Infra Red Quantum Dots (NIR QD’s) to image mesoderm migration in vivo with single cell resolution and provide quantitative in vivo data regarding migration rates. In addition we use QD’s to address the function of the focal adhesion kinase (FAK) in this movement. Inhibition of FAK blocks mesoderm spreading and migration both in vitro and in vivo without affecting convergent extension highlighting the molecular differences between the two movements. These results provide new insights about the role of FAK and of focal adhesions during gastrulation and provide a new tool for the study of morphogenesis in vivo.

Origin of the prechordal plate and patterning of the anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster

We analyzed the notochord formation, formation of the prechordal plate, and patterning of anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster. The lower (LDMZ) and upper (UDMZ) domains of the dorsal marginal zone (DMZ) of the early gastrula involuted and formed two distinct domains: the anterior fore-notochordal endodermal roof and the posterior domain containing the prospective notochord. Cygsc is expressed in the LDMZ from the onset of gastrulation, and the Cygsc-expressing LDMZ planarly induces the notochord in the UDMZ at the early to mid gastrula stages. At the mid to late gastrula stages, part of the Cygsc-expressing LDMZ is confined to the prechordal plate. On the other hand, Cybra expression only begins at mid gastrula stage, coincident with notochord induction at this stage. Anteroposterior regional specificity of the neural plate was patterned by the posterior domain of the involuting archenteron roof containing the prospective notochord at the mid to late gastrula stages. Cynops gastrulation thus differs significantly from Xenopus gastrulation in that the regions of the DMZ are specified from the onset of gastrulation, while the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Thus we propose that Cynops gastrulation is divided into two phases: a notochord induction phase in the early to mid gastrula, and a neural induction phase in the mid to late gastrula.

Role of p21‐activated kinase in cell polarity and directional mesendoderm migration in the Xenopus gastrula

The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility. Using a kinase-dead mutant of xPak1, we show that during Xenopus gastrulation, the kinase activity of Pak1 is required upstream of Cdc42 for the establishment of cell polarity in the migrating mesendoderm. Overactivation of Pak1 function by the expression of constitutively active xPak1 compromises the maintenance of cell polarity, by indirectly inhibiting RhoA function. Inhibition of cell polarization does not affect the migration of single mesendoderm cells. However, Pak1 inhibition interferes with the guidance of mesendoderm migration by directional cues residing in the extracellular matrix of the blastocoel roof, and with mesendoderm translocation in the embryo.

Noncanonical Wnt/PCP Signaling During Vertebrate Gastrulation

The branch of the Wnt pathway, related to planar cell polarity signaling in Drosophila, is fundamental not only to the establishment of tissue polarity but also to a variety of morphogenetic processes in vertebrates. The genetic pathway has been noted for its similarity as well as divergence of between vertebrates and Drosophila. This review focuses on issues related to the complexity of the output of the planar cell polarity pathway during gastrulation in zebrafish and Xenopus and, to a lesser extent, during gastrulation/neurulation in mice.

Oct25 Represses Transcription of Nodal/Activin Target Genes by Interaction with Signal Transducers during Xenopus Gastrulation

The balance between differentiation signals and signals maintaining the undifferentiated state of embryonic cells ensures proper formation of germ layers. The nodal/activin pathway represents one of the major signaling chains responsible for the differentiation of embryonic cells into mesodermal and endodermal germ layers, while Oct4 is one of the major players in the maintenance of an undifferentiated state. Here we show that Oct25, an Oct4 homologue in Xenopus, antagonizes the activity of nodal/activin signaling by inhibiting the transcription of its target genes, Gsc and Mix2. The inhibitory effect is achieved by forming repression complexes on the promoters of Gsc and Mix2 between Oct25 and the signal transducers of the nodal/activin pathway, WBSCR11, FAST1, and Smad2. We have analyzed the significance of the Oct binding site for its inhibitory effect within the Gsc promoter. Albeit VP16-Oct25 fusion protein demonstrated a stimulating effect and EVE-Oct25 revealed a repression effect on an artificial reporter that is composed of eight repeats of Oct binding motifs, both fusions, like wild-type Oct25, inhibited mesendoderm formation and the activity of Gsc and Mix2 promoters. These results suggest that the regulatory effect of Oct25 on the expression of Gsc and Mix2 is mediated by specific protein/protein interactions. Furthermore, we demonstrate that histone deacetylase activities are not required for the inhibitory effect of Oct25. Our results provide a novel view in that Oct25 controls the nodal/activin pathway and thus maintains the undifferentiated state of embryonic cells in preventing them from premature differentiation.

Nucleoredoxin regulates the Wnt/planar cell polarity pathway in Xenopus

The Wnt signaling pathway is conserved across species, and is essential for early development. We previously identified nucleoredoxin (NRX) as a protein that interacts with dishevelled (Dvl) in vivo to negatively regulate the Wnt/beta-catenin pathway. However, whether NRX affects another branch of the Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, remains unclear. Here we show that NRX regulates the Wnt/PCP pathway. In Xenopus laevis, over-expression or depletion of NRX by injection of NRX mRNA or antisense morpholino oligonucleotide, respectively, yields the bent-axis phenotype that is typically observed in embryos with abnormal PCP pathway activity. In co-injection experiments of Dvl and NRX mRNA, NRX suppresses the Dvl-induced bent-axis phenotype. Over-expression or depletion of NRX also suppresses the convergent extension movements that are believed to underlie normal gastrulation. We also found that NRX can inhibit Dvl-induced up-regulation of c-Jun phosphorylation. These results indicate that NRX plays crucial roles in the Wnt/PCP pathway through Dvl and regulates Xenopus gastrulation movements.

PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis

We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin β1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated α5β1 integrin and cytoskeleton strength during cell movement.

Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFβ target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1–3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior–posterior (A–P) patterning, primitive streak formation and left–right (L–R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.

Coordination of Cell Polarity during Xenopus Gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and naïve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity.