Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.     

Wnt/PCP signaling: a veritable polar star in establishing patterns of polarity in embryonic tissues

Embryonic patterning has traditionally been viewed as the establishment of spatially significant gene expression in response to secreted signals. Recent work has highlighted the role of the Wnt/planar cell polarity (PCP) pathway in patterning tissues. Rather than establishing characteristic arrays of gene expression, however, this pathway functions to institute uniform polarity of cells within a tissue. Cells thus polarized can undergo directed migrations, cell divisions, etc., which are essential for normal morphogenesis. In this review, I will highlight the similarities between mechanisms that establish patterns of polarity between Drosophila and vertebrates. Further, I will discuss recent advances with regard to Wnt/PCP signaling in vertebrates.     

Planar cell polarity and a potential role for a Wnt morphogen gradient in stereociliary bundle orientation in the mammalian inner ear

The planar cell polarity (PCP) pathway, a noncanonical Wnt signaling pathway, is crucial for embryonic development in all animals as it is responsible for the regulation of coordinated orientation of structures within the plane of the various epithelia. In the mammalian cochlea, one of the best examples of planar polarity in vertebrates, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are all uniformly polarized. Generation of this polarity is important for hair cell mechanotransduction and auditory perception as stereociliary bundles are only sensitive to vibrations in their single plane of polarization. We describe the two step developmental process that results in the generation of planar polarity in the mammalian inner ear. Furthermore, we review evidence for the role of Wnt signaling, and the possible generation of a Wnt gradient, in planar polarity.     

Compartmentalized Notch signaling sustains epithelial mirror symmetry

Bilateral symmetric tissues must interpret axial references to maintain their global architecture during growth or repair. The regeneration of hair cells in the zebrafish lateral line, for example, forms a vertical midline that bisects the neuromast epithelium into perfect mirror-symmetric plane-polarized halves. Each half contains hair cells of identical planar orientation but opposite to that of the confronting half. The establishment of bilateral symmetry in this organ is poorly understood. Here, we show that hair-cell regeneration is strongly directional along an axis perpendicular to that of epithelial planar polarity. We demonstrate compartmentalized Notch signaling in neuromasts, and show that directional regeneration depends on the development of hair-cell progenitors in polar compartments that have low Notch activity. High-resolution live cell tracking reveals a novel process of planar cell inversions whereby sibling hair cells invert positions immediately after progenitor cytokinesis, demonstrating that oriented progenitor divisions are dispensable for bilateral symmetry. Notwithstanding the invariably directional regeneration, the planar polarization of the epithelium eventually propagates symmetrically because mature hair cells move away from the midline towards the periphery of the neuromast. We conclude that a strongly anisotropic regeneration process that relies on the dynamic stabilization of progenitor identity in permissive polar compartments sustains bilateral symmetry in the lateral line.     

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

Wnt signaling in development and disease

ABSTRACT: Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

Planar cell polarity signaling in craniofacial development

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.     

The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

Planar cell polarity signaling in craniofacial development

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.     

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.     

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.     

Directional cell migration establishes the axes of planar polarity in the posterior lateral-line organ of the zebrafish

The proper orientation of mechanosensory hair cells along the lateral-line organ of a fish or amphibian is essential for the animal's ability to sense directional water movements. Within the sensory epithelium, hair cells are polarized in a stereotyped manner, but the mechanisms that control their alignment relative to the body axes are unknown. We have found, however, that neuromasts can be oriented either parallel or perpendicular to the anteroposterior body axis. By characterizing the strauss mutant zebrafish line and by tracking labeled cells, we have demonstrated that neuromasts of these two orientations originate from, respectively, the first and second primordia. Furthermore, altering the migratory pathway of a primordium reorients a neuromast's axis of planar polarity. We propose that the global orientation of hair cells relative to the body axes is established through an interaction between directional movement by primordial cells and the timing of neuromast maturation.     

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling

The Wnt signaling pathway can be grouped into two classes, the β‐catenin‐dependent and β‐catenin‐independent pathways. Wnt5a signaling through a β‐catenin‐independent pathway promotes microtubule (MT) remodeling during cell‐substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT‐associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus‐ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus‐ends, and depletion of the Kinesin‐1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar‐polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.     

Regulation of microtubules in cell migration

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.     

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.     

Hyphal orientation of Candida albicans is regulated by a calcium-dependent mechanism

Eukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca(2+) cytoplasmic gradient, which is correlated with their ability to exhibit polarized tip growth and to respond to growth-directing extracellular signals. In yeast and other fungi, the polarisome, exocyst, Arp2/3, and Spitzenk?rper protein complexes collectively orchestrate tip growth and cell polarity, but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic Ca(2+) gradients. Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) and imposed electric fields (galvanotropism). The establishment and maintenance of directional growth in relation to these environmental cues was Ca(2+) dependent. Tropisms were attenuated in media containing low Ca(2+), or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca(2+) influx at sites of polarized growth via Ca(2+) channels that are activated by appropriate environmental signals.     

VANG-1 and PRKL-1 cooperate to negatively regulate neurite formation in Caenorhabditis elegans

Neuritogenesis is a critical early step in the development and maturation of neurons and neuronal circuits. While extracellular directional cues are known to specify the site and orientation of nascent neurite formation in vivo, little is known about the genetic pathways that block inappropriate neurite emergence in order to maintain proper neuronal polarity. Here we report that the Caenorhabditis elegans orthologues of Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1), core components of planar cell polarity (PCP) signaling, are required in a subset of peripheral motor neurons to restrict neurite emergence to a specific organ axis. In loss-of-function mutants, neurons display supernumerary neurites that extend inappropriately along the orthogonal anteroposterior (A/P) body axis. We show that autonomous and non-autonomous gene activities are required early and persistently to inhibit the formation or consolidation of growth cone protrusions directed away from organ precursor cells. Furthermore, prkl-1 overexpression is sufficient to suppress neurite formation and reorient neuronal polarity in a vang-1- and dsh-1-dependent manner. Our findings suggest a novel role for a PCP-like pathway in maintaining polarized neuronal morphology by inhibiting neuronal responses to extrinsic or intrinsic cues that would otherwise promote extraneous neurite formation.