Cell shape and epithelial patterning in the Drosophila embryonic epidermis

The development of denticle rows on the ventral Drosophila embryo is a valuable system for studying the genetic control of epithelial patterning. During late embryogenesis, the apical surfaces of denticle-producing cells acquire a distinctive rectangular morphology with long anteroposterior boundaries, along which the denticles form, and short ventrolateral boundaries that stain strongly for adherens junction proteins. We observe that ventrolateral denticle cell boundaries are also convoluted, suggesting that the strong adherens staining results, at least in part, from the additional membrane in these regions. Embryos mutant for the Planar Cell Polarity (PCP) Effector gene multiple wing hairs (mwh), or expressing dominant negative form of the small GTPase Rac1, have cells present between the normal denticle cell rows. These 'Interloper Cells' do not have convoluted ventrolateral boundaries with strong adherens protein staining, but have normal denticle placement, suggesting that adherens protein localization is not critical for denticle cell PCP. Based on these and other observations, we propose that denticle cell morphology arises from an epithelial stretch without junction remodeling. A crude mechanical model suggests that this mechanism can generate both the straight anteroposterior boundaries and the compacted ventrolateral boundaries typical of denticle cells. We discuss the significance of cell adhesion for denticle cell morphogenesis, especially given the established role for Rac1 in cell adhesion.     

Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field

Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the ‘core’ frizzled system, are still the subject of investigation. Examining the deployment of the Ds–Fat system in different tissues of the model organism Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, we examine the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds–Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex.     

Dissecting the PCP pathway: One or more pathways?

Planar cell polarity (PCP), the alignment of cells within 2D tissue planes, involves a set of core molecular regulators highly conserved between animals and cell types. These include the transmembrane proteins Frizzled (Fz) and VanGogh and the cytoplasmic regulators Dishevelled (Dsh) and Prickle. It is widely accepted that this core forms part of a 'PCP pathway' for signal transduction, which can affect cell morphology through activation of an evolutionary ancient regulatory module involving Rho family GTPases and Myosin II, and/or the JNK kinase cascade. We have re-examined the evidence for interactions between the proposed PCP pathway components, and question the placing of the cell morphology regulators in the same pathway as the PCP core. While Fz and Dsh are clearly involved in both PCP and Rho-based cell morphology regulation, available evidence cannot currently discriminate whether these processes are linked mechanistically by a shared Fz/Dsh population, or pass by two distinct pathways.     

Serrate-Notch signaling defines the scope of the initial denticle field by modulating EGFR activation

The Drosophila embryonic epidermis has been a key model for understanding the establishment of cell type diversity across a cellular field. During segmental patterning, distinct signaling territories are established that employ either the Hedgehog, Spitz, Serrate or Wingless ligands. How these pathways control segmental pattern is not completely clear. One major decision occurs as cells are allocated to differentiate either smooth cuticle or denticle type cuticle. This allocation is based on competition between Wingless signaling and Spitz, which activates the Epidermal Growth Factor Receptor (EGFR). Here we show that a main role for Serrate-Notch signaling is to adjust the Spitz signaling domain. Serrate accomplishes this task by activating Notch in a discrete domain, the main purpose of which is to broaden the spatially regulated expression of Rhomboid. This adjusts the breadth of the source for Spitz, since Rhomboid is necessary for the production of active Spitz. We also show that the Serrate antagonist, fringe, must temper Notch activity to insure that the activation of the EGFR is not too robust. Together, Serrate and Fringe modulate Notch activation to generate the proper level of EGFR activation. If Serrate-Notch signaling is absent, the denticle field narrows while the smooth cell field expands, as judged by the expression of the denticle field determinant Ovo/Shaven baby. This establishes one important role for the Serrate signaling territory, which is to define the extent of denticle field specification.     

Epidermal Growth Factor Signalling Controls Myosin II Planar Polarity to Orchestrate Convergent Extension Movements during Drosophila Tubulogenesis

Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis we show that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, we demonstrate that the vector of cell movement is regulated by localised epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality.     

Characterization of the GbpD-activated Rap1 pathway regulating adhesion and cell polarity in Dictyostelium discoideum

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.     

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.     

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.     

Wingless and Hedgehog pattern Drosophila denticle belts by regulating the production of short-range signals

The secreted proteins Wingless and Hedgehog are essential to the elaboration of the denticle pattern in the epidermis of Drosophila embryos. We show that signaling by Wingless and Hedgehog regulates the expression of veinlet (rhomboid) and Serrate, two genes expressed in prospective denticle belts. Thus, Serrate and veinlet (rhom) partake in the last layer of the segmentation cascade. Ultimately, Wingless, Hedgehog, Veinlet (an indirect activator of the Egfr) and Serrate (an activator of Notch) are expressed in non-overlapping narrow stripes. The interface between any two stripes allows a reliable prediction of individual denticle types and polarity suggesting that contact-dependent signaling modulates individual cell fates. Attributes of a morphogen can be ascribed to Hedgehog in this system. However, no single morphogen organises the whole denticle pattern.     

Myosin is involved in postmitotic cell spreading

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.     

Mutual antagonism between signals secreted by adjacent wingless and engrailed cells leads to specification of complementary regions of the Drosophila parasegment.

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. We show that wg mutants have residual mirror-symmetric pattern that is due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern.     

Planar Cell Polarity: Keeping Hairs Straight Is Not So Simple

The fur on a cat''s back, the scales on a fish, or the bristles on a fly are all beautifully organized, with a high degree of polarization in their surface organization. Great progress has been made in understanding how individual cell polarity is established, but our understanding of how cells coordinate their polarity in forming coherent tissues is still fragmentary. The organization of cells in the plane of the epithelium is known as planar cell polarity (PCP), and studies in the past decade have delineated a genetic pathway for the control of PCP. This review will first briefly review data from the Drosophila field, where PCP was first identified and genetically characterized, and then explore how vertebrate tissues become polarized during development.     

Myosin VI plays a role in cell-cell adhesion during epithelial morphogenesis

Myosin VI is an unconventional Myosin that has been implicated in vesicle transport and membrane trafficking. We isolated lethal mutants of Myosin VI, which lack protein once maternal supplies have been utilised during embryogenesis. Dorsal closure, where there is a ring of Myosin VI at the edge of the migrating epithelial sheet, is often abnormal. The sheet of migrating cells is irregular, rather than a smooth epithelium and cells begin to detach. Some embryos hatch into larvae, containing detached cells loose in the haemolymph. Myosin VI is crucial for correct cell morphology and maintenance of adhesive cellular contacts within epithelial cell layers.     

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.     

Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: a ctenophore perspective

ABSTRACT: BACKGROUND: Myosin II (or Myosin Heavy Chain II, MHCII) is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa) and striated muscle cells (MHCIIb). Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. RESULTS: We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus. Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene (PpiMHCIIa) has an expression pattern akin to that of "stem cell markers" (Piwi, Vasa...) and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. CONCLUSION: MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue (PpiMHCIIb2) has retained MHCIIa-like expression features furthermore suggests that muscular expression of the other paralogue, PpiMHCIIb1, was the result of neofunctionalisation within the ctenophore lineage, making independent origin of ctenophore muscle cells a likely option.     

Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis

Abstract

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40?years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.     

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.     

Filament-dependent and -independent localization modes of Drosophila non-muscle myosin II

Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.     

Line up and listen: Planar cell polarity regulation in the mammalian inner ear

The inner ear sensory organs possess extraordinary structural features necessary to conduct mechanosensory transduction for hearing and balance. Their structural beauty has fascinated scientists since the dawn of modern science and ensured a rigorous pursuit of the understanding of mechanotransduction. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity and resolution, and represent perhaps the most distinctive form of a type of cellular polarity, known as planar cell polarity (PCP). Until recently, however, it was not known how the precise PCP of the inner ear sensory organs was achieved during development. Here, we review the PCP of the inner ear and recent advances in the quest for an understanding of its formation.     

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.