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.     

Segment-Specific Adhesion as a Driver of Convergent Extension

Convergent extension, the simultaneous extension and narrowing of tissues, is a crucial event in the formation of the main body axis during embryonic development. It involves processes on multiple scales: the sub-cellular, cellular and tissue level, which interact via explicit or intrinsic feedback mechanisms. Computational modelling studies play an important role in unravelling the multiscale feedbacks underlying convergent extension. Convergent extension usually operates in tissue which has been patterned or is currently being patterned into distinct domains of gene expression. How such tissue patterns are maintained during the large scale tissue movements of convergent extension has thus far not been investigated. Intriguingly, experimental data indicate that in certain cases these tissue patterns may drive convergent extension rather than requiring safeguarding against convergent extension. Here we use a 2D Cellular Potts Model (CPM) of a tissue prepatterned into segments, to show that convergent extension tends to disrupt this pre-existing segmental pattern. However, when cells preferentially adhere to cells of the same segment type, segment integrity is maintained without any reduction in tissue extension. Strikingly, we demonstrate that this segment-specific adhesion is by itself sufficient to drive convergent extension. Convergent extension is enhanced when we endow our in silico cells with persistence of motion, which in vivo would naturally follow from cytoskeletal dynamics. Finally, we extend our model to confirm the generality of our results. We demonstrate a similar effect of differential adhesion on convergent extension in tissues that can only extend in a single direction (as often occurs due to the inertia of the head region of the embryo), and in tissues prepatterned into a sequence of domains resulting in two opposing adhesive gradients, rather than alternating segments.     

Simulating convergent extension by way of anisotropic differential adhesion

Simulations using the Extended Potts Model suggest that anisotropic differential adhesion can account for convergent extension, as observed during embryonic development of the frog Xenopus laevis for example. During gastrulation in these frogs, convergent extension produces longitudinal tissue growth from latitudinal elongation and migration of aligned constituent cells. The Extended Potts Model employs clustered points on a grid to represent subdivided cells with probabilistic displacement of cell boundaries such that small changes in energy drive gradual tissue development. For modeling convergent extension, simulations include anisotropic differential adhesion: the degree of attachment between adjacent elongated cells depends on their relative orientation. Without considering additional mechanisms, simulations based on anisotropic differential adhesion reproduce the hallmark stages of convergent extension in the correct sequence, with random fluctuations as sufficient impetus for cell reorganization.     

Calcium signaling during convergent extension in Xenopus

BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.     

Integrin alpha5beta1 and fibronectin regulate polarized cell protrusions required for Xenopus convergence and extension

BACKGROUND: Integrin recognition of fibronectin is required for normal gastrulation including the mediolateral cell intercalation behaviors that drive convergent extension and the elongation of the frog dorsal axis; however, the cellular and molecular mechanisms involved are unclear. RESULTS: We report that depletion of fibronectin with antisense morpholinos blocks both convergent extension and mediolateral protrusive behaviors in explant preparations. Both chronic depletion of fibronectin and acute disruptions of integrin alpha5beta1 binding to fibronectin increases the frequency and randomizes the orientation of polarized cellular protrusions, suggesting that integrin-fibronectin interactions normally repress frequent random protrusions in favor of fewer mediolaterally oriented ones. In the absence of integrin alpha5beta1 binding to fibronectin, convergence movements still occur but result in convergent thickening instead of convergent extension. CONCLUSIONS: These findings support a role for integrin signaling in regulating the protrusive activity that drives axial extension. We hypothesize that the planar spatial arrangement of the fibrillar fibronectin matrix, which delineates tissue compartments within the embryo, is critical for promoting productive oriented protrusions in intercalating cells.     

Do lamellipodia have the mechanical capacity to drive convergent extension?

Convergent extension (CE), a kinematic motif associated with several important morphogenetic movements in embryos, entails narrowing of a tissue in one in-plane direction and elongation in the other. Although the cell elongation and intercalation which accompany this process have been investigated and relevant genes and biochemical pathways have been studied in multiple organisms, a fundamental question that has not yet been answered is "Do the lamellipodia thought to drive these motions actually have the mechanical capacity to do so?" Here, we address this and a number of related issues using a state-of-the-art computational model which can replicate cell motions, changes in cell shape and tissue deformations. The model is based on the cell-level finite element approach of Chen and Brodland, but has additional features which allow it to model lamellipodium formation and contraction. In studying CE, computational models provide an important complement to molecular approaches because they reveal the "mechanical pathways" through which gene products must ultimately act in order to produce physical movements. The model shows that lamellipodia can drive CE, that they do so through cell intercalations and that the elongated cells characteristic of CE arise only when adjacent tissues resist convergence, a result which we confirm experimentally.     

The planar cell polarity gene strabismus regulates convergence and extension and neural fold closure in Xenopus

We cloned Xenopus Strabismus (Xstbm), a homologue of the Drosophila planar cell or tissue polarity gene. Xstbm encodes four transmembrane domains in its N-terminal half and a PDZ-binding motif in its C-terminal region, a structure similar to Drosophila and mouse homologues. Xstbm is expressed strongly in the deep cells of the anterior neural plate and at lower levels in the posterior notochordal and neural regions during convergent extension. Overexpression of Xstbm inhibits convergent extension of mesodermal and neural tissues, as well as neural tube closure, without direct effects on tissue differentiation. Expression of Xstbm(DeltaPDZ-B), which lacks the PDZ-binding region of Xstbm, inhibits convergent extension when expressed alone but rescues the effect of overexpressing Xstbm, suggesting that Xstbm(DeltaPDZ-B) acts as a dominant negative and that both increase and decrease of Xstbm function from an optimum retards convergence and extension. Recordings show that cells expressing Xstbm or Xstbm(DeltaPDZ-B) fail to acquire the polarized protrusive activity underlying normal cell intercalation during convergent extension of both mesodermal and neural and that this effect is population size-dependent. These results further characterize the role of Xstbm in regulating the cell polarity driving convergence and extension in Xenopus.     

Emergent morphogenesis: Elastic mechanics of a self-deforming tissue

Multicellular organisms are generated by coordinated cell movements during morphogenesis. Convergent extension is a key tissue movement that organizes mesoderm, ectoderm, and endoderm in vertebrate embryos. The goals of researchers studying convergent extension, and morphogenesis in general, include understanding the molecular pathways that control cell identity, establish fields of cell types, and regulate cell behaviors. Cell identity, the size and boundaries of tissues, and the behaviors exhibited by those cells shape the developing embryo; however, there is a fundamental gap between understanding the molecular pathways that control processes within single cells and understanding how cells work together to assemble multicellular structures. Theoretical and experimental biomechanics of embryonic tissues are increasingly being used to bridge that gap. The efforts to map molecular pathways and the mechanical processes underlying morphogenesis are crucial to understanding: (1) the source of birth defects, (2) the formation of tumors and progression of cancer, and (3) basic principles of tissue engineering. In this paper, we first review the process of tissue convergent extension of the vertebrate axis and then review models used to study the self-organizing movements from a mechanical perspective. We conclude by presenting a relatively simple “wedge-model” that exhibits key emergent properties of convergent extension such as the coupling between tissue stiffness, cell intercalation forces, and tissue elongation forces.     

Unravelling the distinct contribution of cell shape changes and cell intercalation to tissue morphogenesis: the case of the Drosophila trachea

Intercalation allows cells to exchange positions in a spatially oriented manner in an array of diverse processes, spanning convergent extension in embryonic gastrulation to the formation of tubular organs. However, given the co-occurrence of cell intercalation and changes in cell shape, it is sometimes difficult to ascertain their respective contribution to morphogenesis. A well-established model to analyse intercalation, particularly in tubular organs, is the Drosophila tracheal system. There, fibroblast growth factor (FGF) signalling at the tip of the dorsal branches generates a ‘pulling’ force believed to promote cell elongation and cell intercalation, which account for the final branch extension. Here, we used a variety of experimental conditions to study the contribution of cell elongation and cell intercalation to morphogenesis and analysed their mutual requirements. We provide evidence that cell intercalation does not require cell elongation and vice versa. We also show that the two cell behaviours are controlled by independent but simultaneous mechanisms, and that cell elongation is sufficient to account for full extension of the dorsal branch, while cell intercalation has a specific role in setting the diameter of this structure. Thus, rather than viewing changes in cell shape and cell intercalation as just redundant events that add robustness to a given morphogenetic process, we find that they can also act by contributing to different features of tissue architecture.     

Neural tube closure requires Dishevelled-dependent convergent extension of the midline

In Xenopus, Dishevelled (Xdsh) signaling is required for both neural tube closure and neural convergent extension, but the connection between these two morphogenetic processes remains unclear. Indeed normal neurulation requires several different cell polarity decisions, any of which may require Xdsh signaling. In this paper we address two issues: (1) which aspects of normal neurulation require Xdsh function; and (2) what role convergent extension plays in the closure of the neural tube. We show that Xdsh signaling is not required for neural fold elevation, medial movement or fusion. Disruption of Xdsh signaling therefore provides a specific tool for uncoupling convergent extension from other processes of neurulation. Using disruption of Xdsh signaling, we demonstrate that convergent extension is crucial to tube closure. Targeted injection revealed that Xdsh function was required specifically in the midline for normal neural tube closure. We suggest that the inherent movement of the neural folds can accomplish only a finite amount of medial progress and that convergent extension of the midline is necessary to reduce the distance between the nascent neural folds, allowing them to meet and fuse. Similar results with Xenopus strabismus implicate the planar cell polarity (PCP) signaling cascade in neural convergent extension and tube closure. Together, these data demonstrate that PCP-mediated convergent extension movements are crucial to proper vertebrate neurulation.     

Requirement of DLG1 for Cardiovascular Development and Tissue Elongation during Cochlear,Enteric, and Skeletal Development: Possible Role in Convergent Extension

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1^-/- mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1^-/- mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1^-/- mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes.     

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.     

The planar polarity gene strabismus regulates convergent extension movements in Xenopus

The signaling mechanisms that specify, guide and coordinate cell behavior during embryonic morphogenesis are poorly understood. We report that a Xenopus homolog of the Drosophila planar cell polarity gene strabismus (stbm) participates in the regulation of convergent extension, a critical morphogenetic process required for the elongation of dorsal structures in vertebrate embryos. Overexpression of Xstbm, which is expressed broadly in early development and subsequently in the nervous system, causes severely shortened trunk structures; a similar phenotype results from inhibiting Xstbm translation using a morpholino antisense oligo. Experiments with Keller explants further demonstrate that Xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue. The specification of dorsal tissues is not affected. The Xstbm phenotype resembles those obtained with several other molecules with roles in planar polarity signaling, including Dishevelled and Frizzled-7 and -8. Unlike these proteins, however, Stbm has little effect on conventional Wnt/beta-catenin signaling in either frog or fly assays. Thus our results strongly support the emerging hypothesis that a vertebrate analog of the planar polarity pathway governs convergent extension movements.     

Bmp activity gradient regulates convergent extension during zebrafish gastrulation

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension.     

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis

During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, we visualized actin and microtubules simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. We cloned full-length XLfc, a microtubule-binding Rho-GEF. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, we believe that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice.     

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.     

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 na?ve 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.