Cell division orientation and planar cell polarity pathways

The orientation of cell division has a crucial role in early embryo body plan specification, axis determination and cell fate diversity generation, as well as in the morphogenesis of tissues and organs. In many instances, cell division orientation is regulated by the planar cell polarity (PCP) pathways: the Wnt/Frizzled non-canonical pathway or the Fat/Dachsous/Four-jointed pathway. Firstly, using asymmetric cell division in both Drosophila and C. elegans, we describe the central role of the Wnt/Frizzled pathway in the regulation of asymmetric cell division orientation, focusing on its cooperation with either the Src kinase pathway or the heterotrimeric G protein pathway. Secondly, we describe our present understanding of the mechanisms by which the planar cell polarity pathways drive tissue morphogenesis by regulating the orientation of symmetric cell division within a field of cells. Finally, we will discuss the important avenues that need to be explored in the future to better understand how planar cell polarity pathways control embryo body plan determination, cell fate specification or tissue morphogenesis by mitotic spindle orientation.     

Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila

Complex organ shapes arise from the coordinate actions of individual cells. The Drosophila egg chamber is an organ-like structure that lengthens along its anterior–posterior axis as it grows. This morphogenesis depends on an unusual form of planar polarity in the organ’s outer epithelial layer, the follicle cells. Interestingly, this epithelium also undergoes a directed migration that causes the egg chamber to rotate around its anterior–posterior axis. However, the functional relationship between planar polarity and migration in this tissue is unknown. We have previously reported that mutations in the Misshapen kinase disrupt follicle cell planar polarity. Here we show that Misshapen’s primary role in this system is to promote individual cell motility. Misshapen decreases integrin levels at the basal surface, which may facilitate detachment of each cell’s trailing edge. These data provide mechanistic insight into Misshapen’s conserved role in cell migration and suggest that follicle cell planar polarity may be an emergent property of individual cell migratory behaviors within the epithelium.     

Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila

Diverse types of epithelial morphogenesis drive development. Similar cytoskeletal and cell adhesion machinery orchestrate these changes, but it is unclear how distinct tissue types are produced. Thus, it is important to define and compare different types of morphogenesis. We investigated cell flattening and elongation in the amnioserosa, a squamous epithelium formed at Drosophila gastrulation. Amnioserosa cells are initially columnar. Remarkably, they flatten and elongate autonomously by perpendicularly rotating the microtubule cytoskeleton--we call this 'rotary cell elongation'. Apical microtubule protrusion appears to initiate the rotation and microtubule inhibition perturbs the process. F-actin restrains and helps orient the microtubule protrusions. As amnioserosa cells elongate, they maintain their original cell-cell contacts and develop planar polarity. Myosin II localizes to anterior-posterior contacts, while the polarity protein Bazooka (PAR-3) localizes to dorsoventral contacts. Genetic analysis revealed that Myosin II and Bazooka cooperate to properly position adherens junctions. These results identify a specific cellular mechanism of squamous tissue morphogenesis and molecular interactions involved.     

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.     

Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase–binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.     

Planar polarity and short-range polarization in Drosophila embryos

Planar cell polarity is a common and probably universal feature of epithelial cells throughout their life. It is not only visible in the external parts of adult animals and plants, but also present in newborn cells such as in the primary Drosophila epithelium. It controls not only cell shape and differentiation, but also cell motility, cell shape changes and it directs how animals are shaped. In this review, we report how planar cell polarity arises in Drosophila embryos and thereby illustrate how general and extensive planar polarity is during development, from the very beginning to the end. We present the main features of planar cell polarization in Drosophila embryos, in particular the fact that it occurs over a short range of just a few cell diameters, and within a very short time window. We contrast these with other systems, such as the adult Drosophila wing where planar cell polarity occurs at longer range.     

Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms

Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the hair cell-intrinsic polarity machinery that establishes the V-shape of the hair bundle is poorly understood. Here, we show that the microtubule motor subunit Kif3a regulates hair cell polarization through both ciliary and non-ciliary mechanisms. Disruption of Kif3a in the inner ear led to absence of the kinocilium, a shortened cochlear duct and flattened hair bundle morphology. Moreover, basal bodies are mispositioned along both the apicobasal and planar polarity axes of mutant hair cells, and hair bundle orientation was uncoupled from the basal body position. We show that a non-ciliary function of Kif3a regulates localized cortical activity of p21-activated kinases (PAK), which in turn controls basal body positioning in hair cells. Our results demonstrate that Kif3a-PAK signaling coordinates planar polarization of the hair bundle and the basal body in hair cells, and establish Kif3a as a key component of the hair cell-intrinsic polarity machinery, which acts in concert with the tissue polarity pathway.     

Ups and downs of tissue and planar polarity in plants

The polar orientation of cells within a tissue is an intensively studied research area in animal cells. The term planar polarity refers to the common polar arrangement of cells within the plane of an epithelium. In plants, the subcellular analysis of tissue polarity has been limited by the lack of appropriate markers. Recently, research on plant tissue polarity has come of age. Advances are based on studies of Arabidopsis patterning, cell polarity and auxin transport mutants employing the coordinated, polar localization of auxin transporters and the planar polarity of root epidermal hairs as markers. These approaches have revealed auxin transport and response, vesicular trafficking, membrane sterol and cytoskeletal requirements of tissue polarity. This review summarizes recent progress in research on vascular tissue and planar epidermal polarity in the Arabidopsis root and compares it to findings on planar polarity in animals and cell polarity in yeast.     

Translating cell polarity into tissue elongation

Planar cell polarity, the orientation of single-cell asymmetries within the plane of a multicellular tissue, is essential to generating the shape and dimensions of organs and organisms. Planar polarity systems align cell behavior with the body axes and orient the cellular processes that lead to tissue elongation. Using Drosophila as a model system, significant progress has been made toward understanding how planar polarity is generated by biochemical and mechanical signals. Recent studies using time-lapse imaging reveal that cells engage in a number of active behaviors whose orientation and dynamics translate planar cell polarity into tissue elongation. Here we review recent progress in understanding the cellular mechanisms that link planar polarity to large-scale changes in tissue structure.     

Principles of planar polarity in animal development

Planar polarity describes the coordinated polarisation of cells or structures in the plane of a tissue. The patterning mechanisms that underlie planar polarity are well characterised in Drosophila, where many events are regulated by two pathways: the 'core' planar polarity complex and the Fat/Dachsous system. Components of both pathways also function in vertebrates and are implicated in diverse morphogenetic processes, some of which self-evidently involve planar polarisation and some of which do not. Here, we review the molecular mechanisms and cellular consequences of planar polarisation in diverse contexts, seeking to identify the common principles across the animal kingdom.     

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior–posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.     

Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine.

A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10 degrees of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.     

Insect epidermis: polarity patterns after grafting result from divergent cell adhesions between host and graft tissue

Insect epidermal cells display planar polarity (i.e. polarity in the plane of the cell sheet) by secreting oriented cuticular denticles and bristles before each moult. We investigate how cell polarities in an abdominal segment are uniformly oriented towards the posterior of the animal. Recently we have shown for the cotton bug Dysdercus that, in 180 degrees-rotated grafts pretreated with colchicine, graft cells tend to adopt the orientation prevailing in surrounding host cells via an intermediate stage with outward oriented denticles (Nübler-Jung and Grau, 1987). Here we show that, in untreated grafts that were transposed along the anteroposterior segment axis, the denticles also always tend to point outwards. This independence of the polarity pattern from the direction of transposition is compatible neither with a gradient model for polarity control, nor with the assumption that epidermal cells orient according to the local sequence of distinctly differentiated cells. Instead we found that outward orientation of graft denticles correlates with an elongation of epidermal cells along a host-graft border with divergent cell adhesiveness. We therefore propose that outward orientation in a graft results from a combination of two factors: epidermal cells stretch along an interface with divergent cell adhesiveness, and they form a denticle perpendicular to their long axis. By analogy, the normal anteroposterior orientation of denticles in a segment may result because epidermal cells tend to elongate parallel to the segment boundary and to form denticles perpendicular to this mediolateral cell elongation, i.e. along the anteroposterior segment axis.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Patterned gene expression directs bipolar planar polarity in Drosophila

During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. Here, we demonstrate that A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement.     

Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.     

Insect epidermis: disturbance of supracellular tissue polarity does not prevent the expression of cell polarity

Summary The insect integument displays planar tissue polarity in the uniform orientation of polarized cuticular structures. In a body segment, for example, the denticles and bristles produced by the constituent epidermal cells point posteriorly. Colchicine can abolish this uniform orientation while still allowing individual cells to form orientated cuticular structures and thereby to express cell polarity. This suggests that an individual cell in a sheet can establish planar polarity without reference to some kind of covert supracellular cue (such as a morphogen gradient) in the epidermis as a whole. The results also indicate that colchicine interferes — directly or indirectly — with the mechanisms involved in aligning the polarity axes of individual cells into a common orientation, thereby generating supracellular or tissue polarity.     

Asymmetric localisation of planar polarity proteins: Mechanisms and consequences

Planar polarisation of tissues is essential for many aspects of developmental patterning. It is regulated by a conserved group of core planar polarity proteins, which localise asymmetrically within cells prior to morphological signs of polarisation. A subset of these core proteins also interact across cell boundaries, mediating intercellular communication that co-ordinates polarity between neighbouring cells. Core protein localisation subsequently mediates changes in the actin cytoskeleton which lead to overt polarisation. In this review we discuss the mechanisms by which the core planar polarity proteins become asymmetrically localised, and the significance of this subcellular localisation for both intercellular communication and downstream effects on the cytoskeleton.