A one-dimensional model of PCP signaling: Polarized cell behavior in the notochord of the ascidian Ciona

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell–cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway’s earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell’s polarity state can be changed and then restored, underscoring the Ciona notochord’s amenability for in vivo studies of PCP.     

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. Results. In the present study, the role of the Wnt5 genes, Hr‐Wnt5α (Halocynthia roretzi Wnt5α) and Hr‐Wnt5β, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64‐cell stage. Hr‐Wnt5α is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5α plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5α indicated that a Wnt5α‐manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5α works in a cell‐autonomous manner. This is further supported by comparison of the results of Wnt5α and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. Conclusion. The present study highlights the role of the Wnt5α signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5α functions in a cell‐autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.     

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.     

Whole-organ cell shape analysis reveals the developmental basis of ascidian notochord taper

Here we use in toto imaging together with computational segmentation and analysis methods to quantify the shape of every cell at multiple stages in the development of a simple organ: the notochord of the ascidian Ciona savignyi. We find that cell shape in the intercalated notochord depends strongly on anterior–posterior (AP) position, with cells in the middle of the notochord consistently wider than cells at the anterior or posterior. This morphological feature of having a tapered notochord is present in many chordates. We find that ascidian notochord taper involves three main mechanisms: Planar Cell Polarity (PCP) pathway-independent sibling cell volume asymmetries that precede notochord cell intercalation; the developmental timing of intercalation, which proceeds from the anterior and posterior towards the middle; and the differential rates of notochord cell narrowing after intercalation. A quantitative model shows how the morphology of an entire developing organ can be controlled by this small set of cellular mechanisms.     

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.     

Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K-->M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical Wnt signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical Wnt signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical Wnt versus PCP signaling cascades.     

Disruption of planar cell polarity activity leads to developmental biliary defects

Planar cell polarity (PCP) establishes polarity within an epithelial sheet. Defects in PCP are associated with developmental defects involving directional cell growth, including defects in kidney tubule elongation that lead to formation of kidney cysts. Given the strong association between kidney cyst formation and developmental biliary defects in patients and in animal models, we investigated the importance of PCP in biliary development. Here we report that in zebrafish, morpholino antisense oligonucleotide-mediated knockdown of PCP genes including prickle-1a (pk1a) led to developmental biliary abnormalities, as well as localization defects of the liver and other digestive organs. The defects in biliary development appear to be mediated via downstream PCP targets such as Rho kinase, Jun kinase (JNK), and both actin and microtubule components of the cytoskeleton. Knockdown of pk1a led to decreased expression of vhnf1, a homeodomain gene previously shown to be involved in biliary development and in kidney cyst formation; forced expression of vhnf1 mRNA led to rescue of the pk1a morphant phenotype. Our results demonstrate that PCP plays an important role in vertebrate biliary development, interacting with other factors known to be involved in biliary morphogenesis.     

Novel regulators of planar cell polarity: a genetic analysis in Drosophila

Planar cell polarity (PCP) is a common feature of many epithelia and epithelial organs. Although progress has been made in the dissection of molecular mechanisms regulating PCP, many questions remain. Here we describe a screen to identify novel PCP regulators in Drosophila. We employed mild gain-of-function (GOF) phenotypes of two cytoplasmic Frizzled (Fz)/PCP core components, Diego (Dgo) and Prickle (Pk), and screened these against the DrosDel genome-wide deficiency collection for dominant modifiers. Positive genomic regions were rescreened and narrowed down with smaller overlapping deficiencies from the Exelixis collection and RNAi-mediated knockdown applied to individual genes. This approach isolated new regulators of PCP, which were confirmed with loss-of-function analyses displaying PCP defects in the eye and/or wing. Furthermore, knockdown of a subset was also sensitive to dgo dosage or dominantly modified a dishevelled (dsh) GOF phenotype, supporting a role in Fz/PCP-mediated polarity establishment. Among the new "PCP" genes we identified several kinases, enzymes required for lipid modification, scaffolding proteins, and genes involved in substrate modification and/or degradation. Interestingly, one of them is a member of the Meckel-Gruber syndrome factors, associated with human ciliopathies, suggesting an important role for cell polarity in nonciliated cells.     

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

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

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

Oriented cell divisions in the extending germband of Drosophila

Tissue elongation is a general feature of morphogenesis. One example is the extension of the germband, which occurs during early embryogenesis in Drosophila. In the anterior part of the embryo, elongation follows from a process of cell intercalation. In this study, we follow cell behaviour at the posterior of the extending germband. We find that, in this region, cell divisions are mostly oriented longitudinally during the fast phase of elongation. Inhibiting cell divisions prevents longitudinal deformation of the posterior region and leads to an overall reduction in the rate and extent of elongation. Thus, as in zebrafish embryos, cell intercalation and oriented cell division together contribute to tissue elongation. We also show that the proportion of longitudinal divisions is reduced when segmental patterning is compromised, as, for example, in even skipped (eve) mutants. Because polarised cell intercalation at the anterior germband also requires segmental patterning, a common polarising cue might be used for both processes. Even though, in fish embryos, both mechanisms require the classical planar cell polarity (PCP) pathway, germband extension and oriented cell divisions proceed normally in embryos lacking dishevelled (dsh), a key component of the PCP pathway. An alternative means of planar polarisation must therefore be at work in the embryonic epidermis.     

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.     

Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.     

Tissue polarity and PCP protein function: C. elegans as an emerging model

Polarity is the basis for the generation of cell diversity, as well as the organization, morphogenesis, and functioning of tissues. Studies in Caenorhabditis elegans have provided much insight into PAR-protein mediated polarity; however, the molecules and mechanisms critical for cell polarization within the plane of epithelia have been identified in other systems. Tissue polarity in C. elegans is organized by Wnt-signaling with some resemblance to the Wnt/planar cell polarity (PCP) pathway, but lacking core PCP protein functions. Nonetheless, recent studies revealed that conserved PCP proteins regulate directed cell migratory events in C. elegans, such as convergent extension movements and neurite formation and guidance. Here, we discuss the latest insights and use of C. elegans as a PCP model.