RESUMEN
Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. Here, we have analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, we found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and is driven by myosin II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.This article has an associated 'The people behind the papers' interview.
Asunto(s)
Drosophila melanogaster/embriología , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Matriz Extracelular/fisiología , Miembro Posterior/embriología , Morfogénesis/fisiología , Animales , Animales Modificados Genéticamente , Membrana Basal/embriología , Membrana Basal/crecimiento & desarrollo , Fenómenos Biomecánicos , Tipificación del Cuerpo/fisiología , Comunicación Celular/fisiología , Proliferación Celular , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Miembro Posterior/crecimiento & desarrollo , Miosina Tipo II/fisiología , Proteolisis , Tensión SuperficialRESUMEN
Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.
Asunto(s)
Apoptosis , Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Células Epiteliales/citología , Epitelio/embriología , Morfogénesis , Uniones Adherentes/química , Uniones Adherentes/metabolismo , Animales , Forma de la Célula , Células Epiteliales/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismoRESUMEN
Apoptosis, or programmed cell death, is a form of cell suicide that is extremely important for ridding the body of cells that are no longer required, to protect the body against hazardous cells, such as cancerous ones, and to promote tissue morphogenesis during animal development. Upon reception of a death stimulus, the doomed cell activates biochemical pathways that eventually converge on the activation of dedicated enzymes, caspases. Numerous pieces of information on the biochemical control of the process have been gathered, from the successive events of caspase activation to the identification of their targets, such as lamins, which constitute the nuclear skeleton. Yet, evidence from multiple systems now shows that apoptosis is also a mechanical process, which may even ultimately impinge on the morphogenesis of the surrounding tissues. This mechanical role relies on dramatic actomyosin cytoskeleton remodelling, and on its coupling with the nucleus before nucleus fragmentation. Here, we provide an overview of apoptosis before describing how apoptotic forces could combine with selective caspase-dependent proteolysis to orchestrate nucleus destruction.
Asunto(s)
Apoptosis/fisiología , Núcleo Celular/metabolismo , Actomiosina/metabolismo , Animales , Caspasas/metabolismo , Muerte Celular/fisiología , Citoesqueleto/metabolismo , Humanos , Morfogénesis/fisiologíaRESUMEN
Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.
Asunto(s)
Apoptosis , Drosophila melanogaster/genética , Microscopía Fluorescente/métodos , Animales , Caspasas/genética , Clonación Molecular , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/químicaRESUMEN
Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.
Asunto(s)
Tipificación del Cuerpo/genética , Sistema Nervioso Central/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Linaje de la Célula/genética , Sistema Nervioso Central/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Mutación , Proteínas Nucleares/química , Estructura Terciaria de Proteína/genética , Factores de Transcripción/química , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
During animal development, tissues and organs are partitioned into compartments that do not intermix. This organizing principle is essential for correct tissue morphogenesis. Given that cell sorting defects during compartmentalization in humans are thought to cause malignant invasion and congenital defects such as cranio-fronto-nasal syndrome, identifying the molecular and cellular mechanisms that keep cells apart at boundaries between compartments is important. In both vertebrates and invertebrates, transcription factors and short-range signalling pathways, such as EPH/Ephrin, Hedgehog, or Notch signalling, govern compartmental cell sorting. However, the mechanisms that mediate cell sorting downstream of these factors have remained elusive for decades. Here, we review recent data gathered in Drosophila that suggest that the generation of cortical tensile forces at compartmental boundaries by the actomyosin cytoskeleton could be a general mechanism that inhibits cell mixing between compartments.
Asunto(s)
Actomiosina/metabolismo , Compartimento Celular , Modelos Biológicos , Animales , Humanos , Transducción de SeñalRESUMEN
The conversion of epithelial cells into mesenchymal ones, through a process known as epithelial-mesenchymal transition (or EMT) is a reversible process involved in critical steps of animal development as early as gastrulation and throughout organogenesis. In pathological conditions such as aggressive cancers, EMT is often associated with increased drug resistance, motility and invasiveness. The characterisation of the upstream signals and main decision takers, such as the EMT-transcription factors, has led to the identification of a core molecular machinery controlling the specification towards EMT. However, the cellular execution steps of this fundamental shift are poorly described, especially in cancerous cells. Here we review our current knowledge regarding the stepwise nature of EMT in model organisms as diverse as sea urchin, Drosophila, zebrafish, mouse or chicken. We focus on the cellular dynamics and mechanics of the transitional stages by which epithelial cells progressively become mesenchymal and leave the epithelium. We gather the currently available pieces of the puzzle, including the overlooked property of EMT cells to produce mechanical forces along their apico-basal axis before detaching from their neighbours. We discuss the interplay between EMT and the surrounding tissue. Finally, we propose a conceptual framework of EMT cell dynamics from the very first hint of epithelial cell reorganisation to the successful exit from the epithelial sheet.
Asunto(s)
Transición Epitelial-Mesenquimal , Pez Cebra , Animales , Células Epiteliales , Epitelio , Gastrulación , RatonesRESUMEN
Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, we show that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. We found that knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, our data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Polaridad Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/citología , Morfogénesis , Miosina Tipo II/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Miosina Tipo II/genéticaAsunto(s)
Apoptosis/fisiología , Morfogénesis/fisiología , Animales , Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Células Epiteliales/citología , Extremidades/crecimiento & desarrollo , Humanos , Mamíferos/embriología , Mamíferos/crecimiento & desarrollo , Metamorfosis Biológica/fisiología , Defectos del Tubo Neural/patología , Pupa , Estrés MecánicoRESUMEN
Unravelling the mechanisms controlling cardiac automatism is critical to our comprehension of heart development and cardiac physiopathology. Despite the extensive characterization of the ionic currents at work in cardiac pacemakers, the precise mechanisms initiating spontaneous rhythmic activity and, particularly, those responsible for the specific control of the pacemaker frequency are still matters of debate and have not been entirely elucidated. By using Drosophila as a model animal to analyze automatic cardiac activity, we have investigated the function of a K+ channel, ORK1 (outwardly rectifying K+ channel-1) in cardiac automatic activity. ORK1 is a two-pore domain K+ (K2P) channel, which belongs to a diverse and highly regulated superfamily of potassium-selective leak channels thought to provide baseline regulation of membrane excitability. Cardiac-specific inactivation of Ork1 led to an increase in heart rhythm. By contrast, when overexpressed, ORK1 completely prevented heart beating. In addition, by recording action potentials, we showed that the level of Ork1 activity sets the cardiac rhythm by controlling the duration of the slow diastolic depolarization phase. Our observations identify a new mechanism for cardiac rhythm control and provide the first demonstration that K2P channels regulate the automatic cardiac activity.
Asunto(s)
Relojes Biológicos/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Contracción Miocárdica/fisiología , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Potenciales de Acción/fisiología , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Estimulación Eléctrica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Frecuencia Cardíaca/fisiología , Hibridación in Situ , Larva/fisiología , Canales de Potasio/genética , ARN Bicatenario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mechanical forces are critical regulators of cell shape changes and developmental morphogenetic processes. Forces generated along the epithelium apico-basal cell axis have recently emerged as essential for tissue remodeling in three dimensions. Yet the cellular machinery underlying those orthogonal forces remains poorly described. We found that during Drosophila leg folding cells eventually committed to die produce apico-basal forces through the formation of a dynamic actomyosin contractile tether connecting the apical surface to a basally relocalized nucleus. We show that the nucleus is anchored to basal adhesions by a basal F-actin network and constitutes an essential component of the force-producing machinery. Finally, we demonstrate force transmission to the apical surface and the basal nucleus by laser ablation. Thus, this work reveals that the nucleus, in addition to its role in genome protection, actively participates in mechanical force production and connects the contractile actomyosin cytoskeleton to basal adhesions.
Asunto(s)
Actinas/metabolismo , Actomiosina/metabolismo , Núcleo Celular/fisiología , Citoesqueleto/fisiología , Drosophila melanogaster/metabolismo , Epitelio/fisiología , Morfogénesis , Animales , Apoptosis , Adhesión Celular , Drosophila melanogaster/crecimiento & desarrollo , Epitelio/crecimiento & desarrollo , Femenino , MasculinoRESUMEN
It is now well established that apoptosis is induced in response to mechanical strain. Indeed, increasing compressive forces induces apoptosis in confined spheroids of tumour cells, whereas releasing stress reduces apoptosis in spheroids cultivated in free suspension (Cheng et al., 2009). Apoptosis can also be induced by applying a 100 to 250MPa pressure, as shown in different cultured cells (for review, see (Frey et al., 2008)). During epithelium development, the pressure caused by a fast-growing clone can trigger apoptosis at the vicinity of the clone, mediating mechanical cell competition (Levayer et al., 2016). While the effect of strain has long been known for its role in apoptosis induction, the reciprocal mechanism has only recently been highlighted. First demonstrated at the cellular level, the effect of an apoptotic cell on its direct neighbours has been analysed in different kinds of monolayer epithelium (Gu et al., 2011; Rosenblatt et al., 2001; Kuipers et al., 2014; Lubkov & Bar-Sagi, 2014). More recently, the concept of a broader impact of apoptotic cell behaviours on tissue mechanical strain has emerged from the characterisation of tissue remodelling during Drosophila development (Toyama et al., 2008; Monier et al., 2015). In the present review, we summarize our current knowledge on the mechanical impact of apoptosis during tissue remodelling.
Asunto(s)
Apoptosis/genética , Drosophila melanogaster/crecimiento & desarrollo , Células Epiteliales/citología , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Abdomen/crecimiento & desarrollo , Animales , División Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Modelos Biológicos , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Estrés MecánicoRESUMEN
Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.
Asunto(s)
Inactivación por Luz Asistida por Cromóforo/métodos , Proteínas de Drosophila/antagonistas & inhibidores , Drosophila melanogaster/genética , Proteínas Fluorescentes Verdes/metabolismo , Miosina Tipo II/antagonistas & inhibidores , Fármacos Fotosensibilizantes/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Recuperación de Fluorescencia tras Fotoblanqueo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Microscopía Confocal , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fármacos Fotosensibilizantes/química , Imagen de Lapso de TiempoRESUMEN
Beyond safeguarding the organism from cell misbehavior and controlling cell number, apoptosis (or programmed cell death) plays key roles during animal development. In particular, it has long been acknowledged that apoptosis participates in tissue remodeling. Yet, until recently, this contribution to morphogenesis was considered as "passive," consisting simply in the local removal of unnecessary cells leading to a new shape. In recent years, applying live imaging methods to study the dynamics of apoptosis in various contexts has considerably modified our vision, revealing that in fact, dying cells remodel their neighborhood actively. Here, we first focus on the intrinsic cellular properties of apoptotic cells during their dismantling, in particular the role of the cytoskeleton during their characteristic morphological changes. Second, we review the various roles of apoptosis during developmental morphogenetic processes and pinpoint the crucial role of live imaging in revealing new concepts, in particular apoptosis as a generator of mechanical forces to control tissue dynamics.
Asunto(s)
Apoptosis/fisiología , Citoesqueleto , Morfogénesis/fisiología , Animales , Forma de la Célula , Drosophila/citología , Drosophila/embriología , Células Epiteliales/citología , Células Epiteliales/fisiología , Extremidades/embriologíaRESUMEN
Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.
Asunto(s)
Actomiosina/metabolismo , Adhesión Celular/fisiología , Compartimento Celular/fisiología , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Animales , Western Blotting , Diferenciación Celular , Movimiento Celular , Citocinesis/fisiología , Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Miosina Tipo II/metabolismoRESUMEN
A functional organ is constituted of diverse cell types. Each one occupies a distinct position and is associated to specific morphological and physiological functions. The identification of the genetic programs controlling these elaborated and highly precise features of organogenesis is crucial to understand how a mature organ works under normal conditions, and how pathologies can develop. Recently, a number of studies have reported a critical role for Hox genes in one example of organogenesis: cardiogenesis in Drosophila. Beyond the interest in understanding the molecular basis of functional cardiogenesis, this system might provide a model for proposing new paradigms of how Hox genes achieve their action throughout development.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Regulación de la Expresión Génica/fisiología , Genes Homeobox/fisiología , AnimalesRESUMEN
In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, we show that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. We characterise the genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. Our results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function.
Asunto(s)
Ecdisona/fisiología , Corazón/crecimiento & desarrollo , Proteínas de Homeodominio/fisiología , Metamorfosis Biológica , Miocitos Cardíacos/citología , Animales , Drosophila , Proteínas de Drosophila/fisiología , Proteínas de Homeodominio/genética , Larva/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The segmented Drosophila linear cardiac tube originates from two cell lineages that give rise to the anterior aorta (AA) and the posterior cardiac tube. The three Hox genes of the Bithorax Complex as well as Antennapedia (Antp) have been shown to be expressed in the posterior cardiac tube, while no Hox gene is expressed in the anterior aorta. We show that the cells of the whole tube adopt the anterior aorta identity in the complete absence of Hox function. Conversely, ectopic expression of Antp, Ultrabithorax (Ubx), or abdominal-A (abd-A) transformed the anterior aorta into posterior cardiac tube by all available criteria, indicating an equivalent early function in their ability to direct a posterior cardiac tube lineage. We further demonstrate that Hox genes act in a subsequent step during cardiac tube organogenesis, specifically on the differentiation of posterior cardiac tube myocytes. In addition, while some of these functions are fulfilled equally well by any one of the three Hox genes, some others are specific to a given Hox. Notably, the gene encoding the anion transporter Na+-Driven Anion Exchanger 1 behaves as a Hox differential transcriptional target and is activated by abd-A in the heart and repressed by Ubx in the posterior aorta. This analysis illustrates the mechanisms by which Hox genes can orchestrate organogenesis and, in particular, allows a clear uncoupling of the different phases of Hox activity in this process.