RESUMO
How complex 3D tissue shape emerges during animal development remains an important open question in biology and biophysics. Here, we discover a mechanism for 3D epithelial shape change based on active, in-plane cellular events that is analogous to inanimate "shape programmable" materials, which undergo blueprinted 3D shape transformations from in-plane gradients of spontaneous strains. We study eversion of the Drosophila wing disc pouch, when the epithelium transforms from a dome into a curved fold, quantifying 3D tissue shape changes and mapping spatial patterns of cellular behaviors on the evolving geometry using cellular topology. Using a physical model inspired by shape programming, we find that active cell rearrangements are the major contributor to pouch eversion and validate this conclusion using a knockdown of MyoVI, which reduces rearrangements and disrupts morphogenesis. This work shows that shape programming is a mechanism for animal tissue morphogenesis and suggests that patterns in nature could present design strategies for shape-programmable materials.
Assuntos
Morfogênese , Asas de Animais , Animais , Asas de Animais/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila , Modelos Biológicos , Discos Imaginais/metabolismo , Discos Imaginais/crescimento & desenvolvimentoRESUMO
How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing of Drosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. Previously, we showed that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned (Etournay et al., 2015). Here, we show that these active cellular behaviors are not guided by the core planar cell polarity (PCP) pathway, a conserved signaling system that guides tissue development in many other contexts. We find no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation they do not significantly affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes during Drosophila pupal wing morphogenesis do not require core PCP as an orientational guiding cue.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Pupa/genética , Asas de Animais/fisiologia , Morfogênese/genética , Polaridade Celular , MutaçãoRESUMO
Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Asas de Animais/metabolismo , Glicólise , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. Here, we describe the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). We show that Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. We therefore uncover a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions.
Assuntos
Proteínas de Drosophila , Somatomedinas , Animais , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Insulina/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Larva/metabolismo , Nutrientes , Somatomedinas/metabolismoRESUMO
Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. Here, we investigate the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, we reveal a pattern of radially oriented cell rearrangements that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, we map tissue stresses and extract key parameters of tissue mechanics. We present a continuum theory showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. Our work reveals a mechanism for the emergence of cellular patterns in morphogenesis.
During development, carefully choreographed cell movements ensure the creation of a healthy organism. To determine their identity and place across a tissue, cells can read gradients of far-reaching signaling molecules called morphogens; in addition, physical forces can play a part in helping cells acquire the right size and shape. Indeed, cells are tightly attached to their neighbors through connections linked to internal components. Structures or proteins inside the cells can pull on these junctions to generate forces that change the physical features of a cell. However, it is poorly understood how these forces create patterns of cell size and shape across a tissue. Here, Dye, Popovic et al. combined experiments with physical models to examine how cells acquire these physical characteristics across the developing wing of fruit fly larvae. This revealed that cells pushing and pulling on one another create forces that trigger internal biochemical reorganization for instance, force-generating structures become asymmetrical. In turn, the cells exert additional forces on their neighbors, setting up a positive feedback loop which results in cells adopting the right size and shape across the organ. As such, cells in the fly wing can spontaneously self-organize through the interplay of mechanical and biochemical signals, without the need for pre-existing morphogen gradients. A refined understanding of how physical forces shape cells and organs would help to grasp how defects can emerge during development. This knowledge would also allow scientists to better grow tissues and organs in the laboratory, both for theoretical research and regenerative medicine.
Assuntos
Forma Celular , Drosophila melanogaster/fisiologia , Discos Imaginais/fisiologia , Mecanotransdução Celular , Asas de Animais/fisiologia , Animais , Padronização Corporal , Divisão Celular , Polaridade Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Retroalimentação Fisiológica , Feminino , Discos Imaginais/embriologia , Masculino , Modelos Biológicos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Estresse Mecânico , Fatores de Tempo , Asas de Animais/embriologiaRESUMO
The levels of nuclear protein Lamin A/C are crucial for nuclear mechanotransduction. Lamin A/C levels are known to scale with tissue stiffness and extracellular matrix levels in mesenchymal tissues. But in epithelial tissues, where cells lack a strong interaction with the extracellular matrix, it is unclear how Lamin A/C is regulated. Here, we show in epithelial tissues that Lamin A/C levels scale with apico-basal cell compression, independent of tissue stiffness. Using genetic perturbations in Drosophila epithelial tissues, we show that apico-basal cell compression regulates the levels of Lamin A/C by deforming the nucleus. Further, in mammalian epithelial cells, we show that nuclear deformation regulates Lamin A/C levels by modulating the levels of phosphorylation of Lamin A/C at Serine 22, a target for Lamin A/C degradation. Taken together, our results reveal a mechanism of Lamin A/C regulation which could provide key insights for understanding nuclear mechanotransduction in epithelial tissues.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Lamina Tipo A/metabolismo , Laminas/metabolismo , Mecanotransdução Celular/fisiologia , Estresse Mecânico , Animais , Linhagem Celular , Cães , Drosophila , Proteínas de Drosophila/genética , Epitélio/metabolismo , Lamina Tipo A/genética , Laminas/genética , Células Madin Darby de Rim Canino , FosforilaçãoRESUMO
Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.
Assuntos
Membrana Celular/metabolismo , Glicólise/genética , Glicólise/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Potenciais da Membrana/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Transporte Biológico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Gramicidina/uso terapêutico , Lipoproteínas , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Receptor Smoothened/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/patologia , Asas de Animais/fisiologiaRESUMO
The signaling protein Sonic Hedgehog (SHH) is crucial for the development and function of many vertebrate tissues. It remains largely unclear, however, what defines the range and specificity of pathway activation. The adrenal gland represents a useful model to address this question, where the SHH pathway is activated in a very specific subset of cells lying near the SHH-producing cells, even though there is an abundance of lipoproteins that would allow SHH to travel and signal long-range. We determine that, whereas adrenal cells can secrete SHH on lipoproteins, this form of SHH is inactive due to the presence of cosecreted inhibitors, potentially explaining the absence of long-range signaling. Instead, we find that SHH-producing cells signal at short range via membrane-bound SHH, only to receiving cells with primary cilia. Finally, our data from NCI-H295R adrenocortical carcinoma cells suggest that adrenocortical tumors may evade these regulatory control mechanisms by acquiring the ability to activate SHH target genes in response to TGF-ß.
Assuntos
Glândulas Suprarrenais/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Cílios/genética , Proteínas Hedgehog/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Studying how epithelia respond to mechanical stresses is key to understanding tissue shape changes during morphogenesis. Here, we study the viscoelastic properties of the Drosophila wing epithelium during pupal morphogenesis by quantifying mechanical stress and cell shape as a function of time. We find a delay of 8 h between maximal tissue stress and maximal cell elongation, indicating a viscoelastic deformation of the tissue. We show that this viscoelastic behavior emerges from the mechanosensitivity of endocytic E-cadherin turnover. The increase in E-cadherin turnover in response to stress is mediated by mechanosensitive relocalization of the E-cadherin binding protein p120-catenin (p120) from cell junctions to cytoplasm. Mechanosensitivity of E-cadherin turnover is lost in p120 mutant wings, where E-cadherin turnover is constitutively high. In this mutant, the relationship between mechanical stress and stress-dependent cell dynamics is altered. Cells in p120 mutant deform and undergo cell rearrangements oriented along the stress axis more rapidly in response to mechanical stress. These changes imply a lower viscosity of wing epithelium. Taken together, our findings reveal that p120-dependent mechanosensitive E-cadherin turnover regulates viscoelastic behavior of epithelial tissues.
Assuntos
Caderinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Mecanotransdução Celular/fisiologia , Animais , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Elasticidade , Epitélio/fisiologia , Masculino , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , ViscosidadeRESUMO
The male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long-term postmating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open-access imaging resource to display our complete findings regarding Rab localization in the accessory gland.
Assuntos
Proteínas de Drosophila/metabolismo , Células Endócrinas/citologia , Fertilidade , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Endócrinas/metabolismo , Genitália Masculina/citologia , Genitália Masculina/metabolismo , Masculino , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.
Assuntos
Drosophila/crescimento & desenvolvimento , Células Epiteliais/citologia , Epitélio/anatomia & histologia , Epitélio/embriologia , Morfogênese , Estresse Mecânico , Actinas/metabolismo , Actomiosina , Amidas/antagonistas & inibidores , Animais , Fenômenos Biomecânicos , Padronização Corporal/genética , Divisão Celular , Proliferação de Células , Forma Celular , Tamanho Celular , Drosophila/anatomia & histologia , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Matriz Extracelular , Discos Imaginais/crescimento & desenvolvimento , Larva/citologia , Larva/metabolismo , Terapia a Laser , Modelos Anatômicos , Modelos Biológicos , Piridinas/antagonistas & inibidoresRESUMO
How cold-blooded animals acclimate to temperature and what determines the limits of their viable temperature range are not understood. Here, we show that Drosophila alter their dietary preference from yeast to plants when temperatures drop below 15°C and that the different lipids present in plants improve survival at low temperatures. We show that Drosophila require dietary unsaturated fatty acids present in plants to adjust membrane fluidity and maintain motor coordination. Feeding on plants extends lifespan and survival for many months at temperatures consistent with overwintering in temperate climates. Thus, physiological alterations caused by a temperature-dependent dietary shift could help Drosophila survive seasonal temperature changes.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Ácidos Graxos Insaturados/metabolismo , Comportamento Alimentar , Lipídeos de Membrana/metabolismo , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Feminino , Fluidez de MembranaRESUMO
Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Drosophila melanogaster/genética , Ecdisterona/farmacologia , Ecdisterona/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes de Insetos , Discos Imaginais/citologia , Discos Imaginais/efeitos dos fármacos , Discos Imaginais/crescimento & desenvolvimento , Insulina/farmacologia , Insulina/fisiologia , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Morfogênese/fisiologia , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Asas de Animais/efeitos dos fármacosRESUMO
Anyone watching a movie of embryonic development immediately appreciates the importance of morphogenetic movements and cell flows that reshape tissue. Dynamic tissue shape changes are genetically choreographed, but their execution is essentially a mechanical event. How the interplay between genetics and tissue mechanics controls tissue shape is a fundamental question. Key insights into this problem have emerged from studies in different model organisms as well as in cultured epithelia. These studies have revealed how gene expression patterns can generate patterns of planar cell polarity that orient cellular force generation and give rise to anisotropic mechanical properties of cells and tissues. These can autonomously bias the rate and orientation of cellular events such as cell divisions, extrusions, neighbor exchanges and shape changes that drive morphogenesis. However recent studies also highlight how autonomously controlled cell dynamics lead to tissue-wide stress patterns framed by mechanical constraints such as cellular connections to extracellular matrices. These stress patterns themselves can orient the cell behaviours underlying morphogenesis. As a result of this interplay, tissue shape emerges in a mechanical process that tightly couples mechanics and genetics.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular , Morfogênese/genética , Animais , Fenômenos Biomecânicos , Divisão Celular , Polaridade Celular , Forma Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Células Epiteliais/citologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Modelos BiológicosRESUMO
MOTIVATION: A significant focus of biological research is to understand the development, organization and function of tissues. A particularly productive area of study is on single layer epithelial tissues in which the adherence junctions of cells form a 2D manifold that is fluorescently labeled. Given the size of the tissue, a microscope must collect a mosaic of overlapping 3D stacks encompassing the stained surface. Downstream interpretation is greatly simplified by preprocessing such a dataset as follows: (i) extracting and mapping the stained manifold in each stack into a single 2D projection plane, (ii) correcting uneven illumination artifacts, (iii) stitching the mosaic planes into a single, large 2D image and (iv) adjusting the contrast. RESULTS: We have developed PreMosa, an efficient, fully automatic pipeline to perform the four preprocessing tasks above resulting in a single 2D image of the stained manifold across which contrast is optimized and illumination is even. Notable features are as follows. First, the 2D projection step employs a specially developed algorithm that actually finds the manifold in the stack based on maximizing contrast, intensity and smoothness. Second, the projection step comes first, implying all subsequent tasks are more rapidly solved in 2D. And last, the mosaic melding employs an algorithm that globally adjusts contrasts amongst the 2D tiles so as to produce a seamless, high-contrast image. We conclude with an evaluation using ground-truth datasets and present results on datasets from Drosophila melanogaster wings and Schmidtae mediterranea ciliary components. AVAILABILITY AND IMPLEMENTATION: PreMosa is available under https://cblasse.github.io/premosa. CONTACT: blasse@mpi-cbg.de or myers@mpi-cbg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Software , Algoritmos , Animais , Artefatos , Cílios/ultraestrutura , Drosophila melanogaster/anatomia & histologia , Platelmintos/ultraestrutura , Asas de Animais/anatomia & histologiaRESUMO
In this article, we propose a general framework to study the dynamics and topology of cellular networks that capture the geometry of cell packings in two-dimensional tissues. Such epithelia undergo large-scale deformation during morphogenesis of a multicellular organism. Large-scale deformations emerge from many individual cellular events such as cell shape changes, cell rearrangements, cell divisions, and cell extrusions. Using a triangle-based representation of cellular network geometry, we obtain an exact decomposition of large-scale material deformation. Interestingly, our approach reveals contributions of correlations between cellular rotations and elongation as well as cellular growth and elongation to tissue deformation. Using this triangle method, we discuss tissue remodeling in the developing pupal wing of the fly Drosophila melanogaster.
Assuntos
Fenômenos Fisiológicos Celulares , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Drosophila melanogaster , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/fisiologiaRESUMO
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
A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells.