RESUMO
Planar cell polarity (PCP) proteins form polarized cortical domains that govern polarity of external structures such as hairs and cilia in both vertebrate and invertebrate epithelia. The mechanisms that globally orient planar polarity are not understood, and are investigated here in the Drosophila wing using a combination of experiment and theory. Planar polarity arises during growth and PCP domains are initially oriented toward the well-characterized organizer regions that control growth and patterning. At pupal stages, the wing hinge contracts, subjecting wing-blade epithelial cells to anisotropic tension in the proximal-distal axis. This results in precise patterns of oriented cell elongation, cell rearrangement and cell division that elongate the blade proximo-distally and realign planar polarity with the proximal-distal axis. Mutation of the atypical Cadherin Dachsous perturbs the global polarity pattern by altering epithelial dynamics. This mechanism utilizes the cellular movements that sculpt tissues to align planar polarity with tissue shape.
Assuntos
Polaridade Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Animais , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Pupa/citologia , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Epithelia are dynamic tissues that self-remodel during their development. During morphogenesis, the tissue-scale organization of epithelia is obtained through a sum of individual contributions of the cells constituting the tissue. Therefore, understanding any morphogenetic event first requires a thorough segmentation of its constituent cells. This task, however, usually involves extensive manual correction, even with semi-automated tools. Here, we present EPySeg, an open-source, coding-free software that uses deep learning to segment membrane-stained epithelial tissues automatically and very efficiently. EPySeg, which comes with a straightforward graphical user interface, can be used as a Python package on a local computer, or on the cloud via Google Colab for users not equipped with deep-learning compatible hardware. By substantially reducing human input in image segmentation, EPySeg accelerates and improves the characterization of epithelial tissues for all developmental biologists.
Assuntos
Epitélio/crescimento & desenvolvimento , Morfogênese/genética , Software , Biologia Computacional , Aprendizado Profundo , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
Epithelial tissues regulate exchanges with the environment. They are highly dynamic and can acquire virtually any shape. At the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the vast number of cells composing an epithelium makes large-scale studies tedious. Here, we present Tissue Analyzer (TA), an open-source tool that can be used to segment epithelia and monitor cell and tissue dynamics.
Assuntos
Processamento de Imagem Assistida por Computador , Epitélio , Processamento de Imagem Assistida por Computador/métodosRESUMO
BACKGROUND: Epithelial junctional networks assume packing geometries characterized by different cell shapes, neighbor number distributions and areas. The development of specific packing geometries is tightly controlled; in the Drosophila wing epithelium, cells convert from an irregular to a hexagonal array shortly before hair formation. Packing geometry is determined by developmental mechanisms that likely control the biophysical properties of cells and their interactions. RESULTS: To understand how physical cellular properties and proliferation determine cell-packing geometries, we use a vertex model for the epithelial junctional network in which cell packing geometries correspond to stable and stationary network configurations. The model takes into account cell elasticity and junctional forces arising from cortical contractility and adhesion. By numerically simulating proliferation, we generate different network morphologies that depend on physical parameters. These networks differ in polygon class distribution, cell area variation, and the rate of T1 and T2 transitions during growth. Comparing theoretical results to observed cell morphologies reveals regions of parameter space where calculated network morphologies match observed ones. We independently estimate parameter values by quantifying network deformations caused by laser ablating individual cell boundaries. CONCLUSIONS: The vertex model accounts qualitatively and quantitatively for the observed packing geometry in the wing disc and its response to perturbation by laser ablation. Epithelial packing geometry is a consequence of both physical cellular properties and the disordering influence of proliferation. The occurrence of T2 transitions during network growth suggests that elimination of cells from the proliferating disc epithelium may be the result of junctional force balances.
Assuntos
Comunicação Celular/fisiologia , Divisão Celular/fisiologia , Proliferação de Células , Células Epiteliais/fisiologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Drosophila , Lasers , Modelos BiológicosRESUMO
The migration of glial chains along the nerve entails directional and coordinated movement. Despite its importance in the formation of the nervous system, this process remains poorly understood, because of the difficulty of manipulating identified cells. Using confocal time-lapse and cell ablation in the whole animal, we provide direct evidence for a discrete number of Drosophila peripheral glial cells acting as pioneers and guiding the rest of the migratory chain. These cells are in direct contact with several follower cells through a very long and stable cytoplasmic extension. The presence of pioneer cells and homotypic interactions at the tip of the chain allows coordinated movement and the formation of a continuous sheath around the nerve. These in vivo data open novel perspectives for understanding the cellular bases of vertebrate glial migration in physiological and pathological conditions.
Assuntos
Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Comunicação Celular/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Fluorescência Verde/biossíntese , Temperatura Alta , Terapia a Laser/métodos , Microscopia Confocal/métodos , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
Morphological diversity is dominated by variation in body proportion [1], which can be described with scaling relationships and mathematical equations, following the pioneering work of D'Arcy Thompson [2] and Julian Huxley [3]. Yet, the cellular processes underlying divergence in size and shape of morphological traits between species remain largely unknown [4-8]. Here, we compare the ovipositors of two related species, Drosophila melanogaster and D. suzukii. D. suzukii has switched its egg-laying niche from rotting to ripe fruit [9]. Along with this shift, the D. suzukii ovipositor has undergone a significant change in size and shape [10]. Using an allometric approach, we find that, while adult ovipositor width has hardly changed between the species, D. suzukii ovipositor length is almost double that of D. melanogaster. We show that this difference mostly arises in a 6-h time window during pupal development. We observe that the developing ovipositors of the two species comprise an almost identical number of cells, with a similar profile of cell shapes and orientations. After cell division stops, we find that the ovipositor area continues to grow in both species through the isotropic expansion of cell apical area and the anisotropic cellular reorganization of the tissue. Remarkably, we find that the lengthening of the D. suzukii ovipositor compared to that of D. melanogaster results from the combination of the accelerated expansion of apical cell size and the enhanced anisotropic rearrangement of cells in the tissue. Therefore, the quantitative fine-tuning of morphogenetic processes can drive evolutionary changes in organ size and shape.
Assuntos
Evolução Biológica , Crescimento Celular , Drosophila/anatomia & histologia , Oviposição , Animais , Drosophila/fisiologia , FemininoRESUMO
Drosophila pupal (P) wing development entails a series of dynamic developmental events, such as epithelial and glial morphogenesis, that are of outstanding interest to cell biologists. Here, we first describe how to prepare P and prepupal (PP) wings for immunofluorescence microscopy. This protocol has been optimized to visualize wing epithelial architecture, such as polarized cortical domains of planar cell polarity proteins. We then provide a protocol to prepare pupae for whole mount live imaging of P wings. This procedure has allowed us to live-image glial cell migration and proliferation along wing sensory nerves.
Assuntos
Biologia do Desenvolvimento/métodos , Drosophila melanogaster/fisiologia , Microscopia de Fluorescência/métodos , Animais , Padronização Corporal , Movimento Celular , Polaridade Celular , Células Epiteliais/citologia , Genes de Insetos , Metamorfose Biológica , Microscopia de Fluorescência/instrumentação , Morfogênese , Neuroglia/citologia , Neurônios Aferentes/metabolismo , Pupa/metabolismo , Asas de Animais/embriologiaRESUMO
The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. Here we provide a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. We show that the adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, we report that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, we demonstrate that planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, our work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Olho/citologia , Olho/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismoRESUMO
Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.
Assuntos
Células Epiteliais/ultraestrutura , Epitélio/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Junções Intercelulares/ultraestrutura , Reconhecimento Automatizado de Padrão/métodos , Software , Animais , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Rastreamento de Células/métodos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Células Epiteliais/metabolismo , Epitélio/metabolismo , Expressão Gênica , Junções Intercelulares/metabolismo , Morfogênese/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Segmentation and tracking of cells in long-term time-lapse experiments has emerged as a powerful method to understand how tissue shape changes emerge from the complex choreography of constituent cells. However, methods to store and interrogate the large datasets produced by these experiments are not widely available. Furthermore, recently developed methods for relating tissue shape changes to cell dynamics have not yet been widely applied by biologists because of their technical complexity. We therefore developed a database format that stores cellular connectivity and geometry information of deforming epithelial tissues, and computational tools to interrogate it and perform multi-scale analysis of morphogenesis. We provide tutorials for this computational framework, called TissueMiner, and demonstrate its capabilities by comparing cell and tissue dynamics in vein and inter-vein subregions of the Drosophila pupal wing. These analyses reveal an unexpected role for convergent extension in shaping wing veins.
Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Epitélio/fisiologia , Morfogênese , Animais , Drosophila/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Imagem com Lapso de TempoRESUMO
How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing.
Assuntos
Drosophila/embriologia , Células Epiteliais/fisiologia , Epitélio/fisiologia , Asas de Animais/embriologia , Animais , Fenômenos Biofísicos , Drosophila/crescimento & desenvolvimento , Modelos Biológicos , Pupa/crescimento & desenvolvimentoRESUMO
Mechanical forces play important roles during tissue organization in developing animals. Many tissues are organized into adjacent, nonmixing groups of cells termed compartments. Boundaries between compartments display a straight morphology and are associated with signaling centers that are important for tissue growth and patterning. Local increases in mechanical tension at cell junctions along compartment boundaries have recently been shown to prevent cell mixing and to maintain straight boundaries. The cellular mechanisms by which local increases in mechanical tension prevent cell mixing at compartment boundaries, however, remain poorly understood. Here, we have used live imaging and quantitative image analysis to determine cellular dynamics at and near the anteroposterior compartment boundaries of the Drosophila pupal abdominal epidermis. We show that cell mixing within compartments involves multiple cell intercalations. Frequency and orientation of cell intercalations are unchanged along the compartment boundaries; rather, an asymmetry in the shrinkage of junctions during intercalation is biased, resulting in cell rearrangements that suppress cell mixing. Simulations of tissue growth show that local increases in mechanical tension can account for this bias in junctional shrinkage. We conclude that local increases in mechanical tension maintain cell populations separate by influencing junctional rearrangements during cell intercalation.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Transdução de Sinais , Abdome/crescimento & desenvolvimento , Animais , Células Epidérmicas , Epiderme/crescimento & desenvolvimento , Processamento de Imagem Assistida por Computador , Pupa/citologia , Pupa/crescimento & desenvolvimento , Estresse MecânicoRESUMO
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.
Assuntos
Polaridade Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Genes Controladores do Desenvolvimento , Larva/citologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Morfogênese , Pupa/citologia , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Asas de Animais/citologia , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Collective cell polarization is an important characteristic of tissues. Epithelia commonly display cellular structures that are polarized within the plane of the tissue. Establishment of this planar cell polarity requires mechanisms that locally align polarized structures between neighbouring cells, as well as cues that provide global information about alignment relative to an axis of a tissue. In the Drosophila ovary, the cadherin Fat2 is required to orient actin filaments located at the basal side of follicle cells perpendicular to the long axis of the egg chamber. The mechanisms directing this orientation of actin filaments, however, remain unknown. Here we show, using genetic mosaic analysis, that fat2 is not essential for the local alignment of actin filaments between neighbouring cells. Moreover, we provide evidence that Fat2 is involved in the propagation of a cue specifying the orientation of actin filaments relative to the tissue axis. Monte Carlo simulations of actin filament orientation resemble the results of the genetic mosaic analysis, if it is assumed that a polarity signal can propagate from a signal source only through a connected chain of wild-type cells. Our results suggest that Fat2 is required for propagating global polarity information within the follicle epithelium through direct cell-cell contact. Our computational model might be more generally applicable to study collective cell polarization in tissues.
Assuntos
Actinas/fisiologia , Caderinas/fisiologia , Polaridade Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Epitélio/fisiologia , Modelos Biológicos , Animais , Simulação por Computador , Método de Monte Carlo , MutaçãoRESUMO
Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.
Assuntos
Técnicas de Cultura de Células/métodos , Biologia do Desenvolvimento/métodos , Lasers , Microscopia Confocal/métodos , Raios Ultravioleta , Animais , Animais Geneticamente Modificados , Comunicação Celular , Biologia do Desenvolvimento/instrumentação , Drosophila , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia Confocal/instrumentaçãoRESUMO
Migration and proliferation have been mostly explored in culture systems or fixed preparations. We present a simple genetic model, the chains of glia moving along fly wing nerves, to follow such dynamic processes by time-lapse in the whole animal. We show that glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed to us define the role of specific cell-cell interactions. First, neurone-glia interactions are not necessary for glia motility but do affect the direction of migration. Second, repulsive interactions between glia control the extent of movement. Finally, autonomous cues control proliferation.