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.     

Integrin-linked Kinase Interactions with ELMO2 Modulate Cell Polarity

Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK–ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG–ELMO2–ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity.     

Formation and functions of asymmetric microtubule organization in polarized cells

Although microtubules are known to be essential for chromosome segregation during cell division, they also play important roles in the regulation and function of cell polarity. Cell polarization is fundamental to appropriate tissue patterning and the regulation of cellular diversity during animal development. In polarized cells, microtubules are often organized asymmetrically along the polarity axis. Recent studies show that such asymmetry in microtubule organization is important to connect a cell's polarization with its polarized functions. In some cases, asymmetrically organized microtubule arrays themselves induce cell polarity. Here we present an overview of the mechanisms and functions of asymmetric microtubule organization and discuss the possible role of microtubule asymmetry in the symmetry-breaking that leads to cell polarization.     

The distribution of Dishevelled in convergently extending mesoderm

Convergent extension (CE) is a conserved morphogenetic movement that drives axial lengthening of the primary body axis and depends on the planar cell polarity (PCP) pathway. In Drosophila epithelia, a polarised subcellular accumulation of PCP core components, such as Dishevelled (Dvl) protein, is associated with PCP function. Dvl has long been thought to accumulate in the mediolateral protrusions in Xenopus chordamesoderm cells undergoing CE. Here we present a quantitative analysis of Dvl intracellular localisation in Xenopus chordamesoderm cells. We find that, surprisingly, accumulations previously observed at mediolateral protrusions of chordamesodermal cells are not protrusion-specific but reflect yolk-free cytoplasm and are quantitatively matched by the distribution of the cytoplasm-filling lineage marker dextran. However, separating cell cortex-associated from bulk Dvl signal reveals a statistical enrichment of Dvl in notochord–somite boundary-(NSB)-directed protrusions, which is dependent upon NSB proximity. Dvl puncta were also observed, but only upon elevated overexpression. These puncta showed no statistically significant spatial bias, in contrast to the strongly posteriorly-enriched GFP-Dvl puncta previously reported in zebrafish. We propose that Dvl distribution is more subtle and dynamic than previously appreciated and that in vertebrate mesoderm it reflects processes other than protrusion as such.     

XGAP, an ArfGAP, is required for polarized localization of PAR proteins and cell polarity in Xenopus gastrulation

To dissect the molecular mechanisms underlying convergent extension (CE), a prominent set of cell movements during Xenopus gastrulation, we performed a functional expression screen and identified a GTPase-activating protein for ADP ribosylation factors (ArfGAP), which we termed XGAP. We demonstrated that XGAP is required to confine or restrict the cellular protrusive activity to the mediolateral ends of cells, where XGAP is normally localized, and therefore for the proper intercalation of cells participating in CE. We also demonstrated that a C-terminal conserved domain of XGAP, but not its GAP activity, is required and sufficient for this intracellular localization and function. We further showed that XGAP physically interacts with the known polarity proteins 14-3-3epsilon, aPKC, and PAR-6 and directs them to the mediolateral ends of dorsal mesoderm cells during gastrulation. We propose that XGAP controls CE through the restriction and maintenance of partitioning-defective (PAR) proteins in the regions that harbor protrusive activity.     

The serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.     

Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation?

Many types of cell show different aspects of polarization. Epithelial cells display a ubiquitous apical-basolateral polarity but often are also polarized in the plane of the epithelium - a feature referred to as 'planar cell polarity' (PCP). In Drosophila all adult epithelial cuticular structures are polarized within the plane, whereas in vertebrates examples of PCP include aspects of skin development, features of the inner ear epithelium, and the morphology and behavior of mesenchymal cells undergoing the morphogenetic movement called 'convergent extension'. Recent advances in the study of PCP establishment are beginning to unravel the molecular mechanisms that underlie this aspect of cell and tissue differentiation. Here I discuss new developments in our molecular understanding of PCP in Drosophila and compare them towhat is known about the regulation of convergent extension in vertebrates.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Gradients and the Specification of Planar Polarity in the Insect Cuticle

In addition to specifying cell fate, there is a wealth of evidence that molecular gradients are also primarily responsible for specifying cell polarity, particularly in the plane of epithelial sheets (“planar polarity”). The first compelling evidence of a role for gradients in specifying planar polarity came from transplantation experiments in the insect cuticle. More recent molecular genetic analyses in the fruit fly Drosophila have begun to give insights into the molecular nature of the gradients involved, and how they are interpreted at the cellular level.Development requires the coordinated specification of at least three attributes: cell fate, tissue size, and cell polarity. In both theory and practice, all three can be specified by the action of gradients. This article examines the experimental evidence for gradients acting to specify cell polarity in developing tissues, considers the mechanisms by which they are thought to act, and discusses what remains unknown. The problem of how cell polarity is specified in the plane of a tissue (“planar polarity”) is addressed. The tissues discussed are all formed from epithelial sheets that also show apicobasal cell polarity.For more than half a century, the preeminent system for studying the regulation of planar polarity in epithelia has been the insect cuticle. This lends itself to the study of the problem by virtue of often being adorned by structures such as hairs, scales, ridges, or other protrusions that reveal the polarity of the underlying cells. However, the lack of polarized structures on the surface of other epithelial-derived tissues should not be taken as evidence that the cells are not planar polarized, because often such polarity is cryptically expressed and only becomes apparent when the cells participate in a polarized process, such as cell division or cell intercalation.     

Actin and microtubules drive differential aspects of planar cell polarity in multiciliated cells

Planar cell polarization represents the ability of cells to orient within the plane of a tissue orthogonal to the apical basal axis. The proper polarized function of multiciliated cells requires the coordination of cilia spacing and cilia polarity as well as the timing of cilia beating during metachronal synchrony. The planar cell polarity pathway and hydrodynamic forces have been shown to instruct cilia polarity. In this paper, we show how intracellular effectors interpret polarity to organize cellular morphology in accordance with asymmetric cellular function. We observe that both cellular actin and microtubule networks undergo drastic reorganization, providing differential roles during the polarized organization of cilia. Using computational angular correlation analysis of cilia orientation, we report a graded cellular organization downstream of cell polarity cues. Actin dynamics are required for proper cilia spacing, global coordination of cilia polarity, and coordination of metachronic cilia beating, whereas cytoplasmic microtubule dynamics are required for local coordination of polarity between neighboring cilia.     

Multiple Wnts redundantly control polarity orientation in Caenorhabditis elegans epithelial stem cells

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1-V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted--the stem cells are not polarized and divide symmetrically--suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals.     

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation

Background

The notochord is a signaling center required for the patterning of the vertebrate embryic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood.

Methodology/Principal Findings

Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete.

Conclusions/Significance

Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.     

Chongmague reveals an essential role for laminin-mediated boundary formation in chordate convergence and extension movements

Although cell intercalation driven by non-canonical Wnt/planar cell polarity (PCP) pathway-dependent mediolateral cell polarity is important for notochord morphogenesis, it is likely that multiple mechanisms shape the notochord as it converges and extends. Here we show that the recessive short-tailed Ciona savignyi mutation chongmague (chm) has a novel defect in the formation of a morphological boundary around the developing notochord. chm notochord cells initiate intercalation normally, but then fail to maintain their polarized cell morphology and migrate inappropriately to become dispersed in the larval tail. This is unlike aimless (aim), a mutation in the PCP pathway component Prickle, which has a severe defect in early mediolateral intercalation but forms a robust notochord boundary. Positional cloning identifies chm as a mutation in the C. savignyi ortholog of the vertebrate alpha 3/4/5 family of laminins. Cs-lamalpha3/4/5 is highly expressed in the developing notochord, and Cs-lamalpha3/4/5 protein is specifically localized to the outer border of the notochord. Notochord convergence and extension, reduced but not absent in both chm and aim, are essentially abolished in the aim/aim; chm/chm double mutant, indicating that laminin-mediated boundary formation and PCP-dependent mediolateral intercalation are each able to drive a remarkable degree of tail morphogenesis in the absence of the other. These mechanisms therefore initially act in parallel, but we also find that PCP signaling has an important later role in maintaining the perinotochordal/intranotochordal polarity of Cs-lamalpha3/4/5 localization.     

Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.     

Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.     

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.     

Apical-basal polarity, Wnt signaling and vertebrate organogenesis

Wnt proteins elicit several distinct signal transduction cascades and regulate multiple cellular processes that have proven essential for embryonic development in all metazoans investigated. During embryonic development, epithelial cells become polarized along two axes: apical/basal and within the plane of the tissue. Growing evidence suggests that polarization along each axis is essential for normal embryonic development and that this polarization is regulated in part by the different branches of the Wnt pathway. Here, we review the role of A/B cell polarity in vertebrate organogenesis with a focus on the involvement of canonical Wnt signaling in this process.     

Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC.     

Essential role for beta-arrestin 2 in the regulation of Xenopus convergent extension movements

beta-Arrestin 2 (betaarr2) is a multifunctional protein that regulates numerous aspects of G-protein-coupled receptor function. However, its possible involvement in developmental processes is poorly understood. In this work, we examined the potential role of betaarr2 during Xenopus early development. Gain- and loss-of-function studies showed that Xenopus betaarr2 (xbetaarr2) is required for proper convergent extension (CE) movements, and normal cell polarization and intercalation without affecting cell fate. Moreover, for CE movements, betaarr2 acts as an essential regulator of dishevelled-mediated PCP (planar cell polarity) signaling, but not G-protein-mediated Ca(2+) signaling. Notably, xbetaarr2 is localized with the same distribution as the dishevelled protein, which is reasonable, as xbetaarr2 is required for dishevelled activation of RhoA. Furthermore, xbetaarr2 interacts with the N-terminal quarter of Daam1 and RhoA proteins, but not Rac1, and regulates RhoA activation through Daam1 activation for CE movements. We provide evidence that the endocytic activity of xbetaarr2 is essential for control of CE movements. Taken together, our results suggest that betaarr2 has a pivotal role in the regulation of Xenopus CE movements.