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1.
BMC Biol ; 20(1): 183, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35999534

RESUMO

BACKGROUND: Efficient tools allowing the extraction of 2D surfaces from 3D-microscopy data are essential for studies aiming to decipher the complex cellular choreography through which epithelium morphogenesis takes place during development. Most existing methods allow for the extraction of a single and smooth manifold of sufficiently high signal intensity and contrast, and usually fail when the surface of interest has a rough topography or when its localization is hampered by other surrounding structures of higher contrast. Multiple surface segmentation entails laborious manual annotations of the various surfaces separately. RESULTS: As automating this task is critical in studies involving tissue-tissue or tissue-matrix interaction, we developed the Zellige software, which allows the extraction of a non-prescribed number of surfaces of varying inclination, contrast, and texture from a 3D image. The tool requires the adjustment of a small set of control parameters, for which we provide an intuitive interface implemented as a Fiji plugin. CONCLUSIONS: As a proof of principle of the versatility of Zellige, we demonstrate its performance and robustness on synthetic images and on four different types of biological samples, covering a wide range of biological contexts.


Assuntos
Algoritmos , Microscopia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Software
2.
Development ; 144(23): 4406-4421, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038308

RESUMO

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 fármacos
3.
Bioinformatics ; 35(15): 2690-2691, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576403

RESUMO

SUMMARY: Using adequate DNA barcodes is essential to unambiguously identify each DNA library within a multiplexed set of libraries sequenced using next-generation sequencers. We introduce DNABarcodeCompatibility, an R-package that allows one to design single or dual-barcoding multiplex experiments by imposing desired constraints on the barcodes (including sequencer chemistry, barcode pairwise minimal distance and nucleotide content), while optimizing barcode frequency usage, thereby allowing one to both facilitate the demultiplexing step and spare expensive library-preparation kits. The package comes with a user-friendly interface and a web app developed in Java and Shiny (https://dnabarcodecompatibility.pasteur.fr), respectively, with the aim to help bridge the expertise of core facilities with the experimental needs of non-experienced users. AVAILABILITY AND IMPLEMENTATION: DNABarcodeCompatibility can be easily extended to fulfil specific project needs. The source codes of the R-package and its user interfaces are publicly available along with documentation at [https://github.com/comoto-pasteur-fr] under the GPL-2 licence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Software , Sequência de Bases , DNA , Biblioteca Gênica , Análise de Sequência
4.
Bioinformatics ; 33(16): 2563-2569, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28383656

RESUMO

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 & histologia
5.
PLoS One ; 19(7): e0305742, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028743

RESUMO

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.


Assuntos
Dependovirus , Orelha Interna , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Dependovirus/genética , Camundongos , Orelha Interna/metabolismo , Orelha Interna/embriologia , Orelha Interna/citologia , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Feminino , Transdução Genética/métodos , Gravidez , Terapia Genética/métodos , Humanos
6.
Development ; 137(8): 1373-83, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20332152

RESUMO

Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.


Assuntos
Cóclea/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Animais , Cílios/fisiologia , Cílios/ultraestrutura , Cóclea/anatomia & histologia , Cóclea/inervação , Cóclea/ultraestrutura , Orelha Interna/citologia , Cabras , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/ultraestrutura , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Neurônios/citologia , Neurônios/fisiologia , Órgão Espiral/fisiologia , Órgão Espiral/ultraestrutura
7.
J Cell Biol ; 222(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-37930352

RESUMO

Although mutations in the SCRIB gene lead to multiple morphological organ defects in vertebrates, the molecular pathway linking SCRIB to organ shape anomalies remains elusive. Here, we study the impact of SCRIB-targeted gene mutations during the formation of the gut epithelium in an organ-on-chip model. We show that SCRIB KO gut-like epithelia are flatter with reduced exposed surface area. Cell differentiation on filters further shows that SCRIB plays a critical role in the control of apical cell shape, as well as in the basoapical polarization of myosin light chain localization and activity. Finally, we show that SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 and identify an evolutionary conserved SHROOM binding site in the SCRIB carboxy-terminal that is required for SCRIB function in the control of apical cell shape. Our results demonstrate that SCRIB plays a key role in epithelial morphogenesis by controlling the epithelial apical contractility during cell differentiation.


Assuntos
Diferenciação Celular , Epitélio , Proteínas de Membrana , Animais , Sítios de Ligação , Evolução Biológica , Forma Celular , Epitélio/crescimento & desenvolvimento , Sistemas Microfisiológicos , Proteínas de Membrana/fisiologia , Morfogênese
8.
Nat Commun ; 12(1): 4354, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272374

RESUMO

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.


Assuntos
COVID-19/patologia , Cílios/ultraestrutura , Depuração Mucociliar/fisiologia , SARS-CoV-2 , Animais , Axonema , Corpos Basais , Cílios/metabolismo , Cílios/patologia , Cricetinae , Citocinas , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Pulmão/patologia , Masculino , Mesocricetus , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Replicação Viral
9.
Phys Rev E ; 95(3-1): 032401, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28415200

RESUMO

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/fisiologia
11.
Elife ; 52016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27228153

RESUMO

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 Tempo
12.
Elife ; 4: e07090, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26102528

RESUMO

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 & desenvolvimento
13.
Curr Biol ; 24(18): 2111-2123, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-25201685

RESUMO

BACKGROUND: The conserved Fat and Core planar cell polarity (PCP) pathways work together to specify tissue-wide orientation of hairs and ridges in the Drosophila wing. Their components form intracellularly polarized complexes at adherens junctions that couple the polarity of adjacent cells and form global patterns. How Fat and Core PCP systems interact is not understood. Some studies suggest that Fat PCP directly orients patterns formed by Core PCP components. Others implicate oriented tissue remodeling in specifying Core PCP patterns. RESULTS: We use genetics, quantitative image analysis, and physical modeling to study Fat and Core PCP interactions during wing development. We show their patterns change during morphogenesis, undergoing phases of coupling and uncoupling that are regulated by antagonistic Core PCP protein isoforms Prickle and Spiny-legs. Evolving patterns of Core PCP are hysteretic: the early Core PCP pattern is modified by tissue flows and then by coupling to Fat PCP, producing sequential patterns that guide hairs and then ridges. Our data quantitatively account for altered hair and ridge polarity patterns in PCP mutants. Premature coupling between Fat and Core PCP explains altered polarity patterns in pk mutants. In other Core PCP mutants, hair polarity patterns are guided directly by Fat PCP. When both systems fail, hairs still align locally and obey signals associated with veins. CONCLUSIONS: Temporally regulated coupling between the Fat and Core PCP systems enables a single tissue to develop sequential polarity patterns that orient distinct morphological structures.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/genética , Transdução de Sinais , Asas de Animais/fisiologia , Animais , Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas com Domínio LIM/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Morfogênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Asas de Animais/crescimento & desenvolvimento
14.
J Cell Sci ; 120(Pt 16): 2838-50, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17666436

RESUMO

Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.


Assuntos
Actinas/metabolismo , Dineínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Cães , Estruturas Embrionárias/citologia , Estruturas Embrionárias/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Miosina VIIa , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Retina/citologia , Retina/metabolismo , Proteína da Zônula de Oclusão-1
15.
J Cell Sci ; 118(Pt 13): 2891-9, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15976448

RESUMO

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.


Assuntos
Dineínas/metabolismo , Células Ciliadas Auditivas Internas/química , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Animais , Linhagem Celular , Dineínas/genética , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Miosina Tipo I , Miosina VIIa , Miosinas/genética
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