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2.
Development ; 146(22)2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31645357

RESUMO

During branching morphogenesis, a simple cluster of cells proliferates and branches to generate an arborized network that facilitates fluid flow. The overall architecture of the mouse lung is established by domain branching, wherein new branches form laterally off the side of an existing branch. The airway epithelium develops concomitantly with a layer of smooth muscle that is derived from the embryonic mesenchyme. Here, we examined the role of smooth muscle differentiation in shaping emerging domain branches. We found that the position and morphology of domain branches are highly stereotyped, as is the pattern of smooth muscle that differentiates around the base of each branch. Perturbing the pattern of smooth muscle differentiation genetically or pharmacologically causes abnormal domain branching. Loss of smooth muscle results in ectopic branching and decreases branch stereotypy. Increased smooth muscle suppresses branch initiation and extension. Computational modeling revealed that epithelial proliferation is insufficient to generate domain branches and that smooth muscle wrapping is required to shape the epithelium into a branch. Our work sheds light on the physical mechanisms of branching morphogenesis in the mouse lung.

3.
Development ; 146(16)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31371376

RESUMO

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

4.
Soft Matter ; 15(28): 5728-5738, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31267114

RESUMO

Type I collagen self-assembles into three-dimensional (3D) fibrous networks. These dynamic viscoelastic materials can be remodeled in response to mechanical and chemical signals to form anisotropic networks, the structure of which influences tissue development, homeostasis, and disease progression. Conventional approaches for fabricating anisotropic networks of type I collagen are often limited to unidirectional fiber alignment over small areas. Here, we describe a new approach for engineering cell-laden networks of aligned type I collagen fibers using 3D microextrusion printing of a collagen-Matrigel ink. We demonstrate hierarchical control of 3D-printed collagen with the ability to spatially pattern collagen fiber alignment and geometry. Our data suggest that collagen alignment results from a combination of molecular crowding in the ink and shear and extensional flows present during 3D printing. We demonstrate that human breast cancer cells cultured on 3D-printed collagen constructs orient along the direction of collagen fiber alignment. We also demonstrate the ability to simultaneously bioprint epithelial cell clusters and control the alignment and geometry of collagen fibers surrounding cells in the bioink. The resulting cell-laden constructs consist of epithelial cell clusters fully embedded in aligned networks of collagen fibers. Such 3D-printed constructs can be used for studies of developmental biology, tissue engineering, and regenerative medicine.

5.
Mol Biol Cell ; 30(10): 1129-1137, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034354

RESUMO

Organoids derived from stem cells or tissues in culture can develop into structures that resemble the in vivo anatomy and physiology of intact organs. Human organoid cultures provide the potential to study human development and model disease processes with the same scrutiny and depth of analysis customary for research with nonhuman model organisms. Resembling the complexity of the actual tissue or organ, patient-derived human organoid studies may accelerate medical research, creating new opportunities for tissue engineering and regenerative medicine, generating knowledge and tools for preclinical studies, including drug development and testing. Biologists are drawn to this system as a new "model organism" to study complex disease phenotypes and genetic variability among individuals using patient-derived tissues. The American Society for Cell Biology convened a task force to report on the potential, challenges, and limitations for human organoid research. The task force suggests ways to ease the entry for new researchers into the field and how to facilitate broader use of this new model organism within the research community. This includes guidelines for reproducibility, culturing, sharing of patient materials, patient consent, training, and communication with the public.


Assuntos
Organoides/metabolismo , Organoides/fisiologia , Animais , Pesquisa Biomédica , Técnicas de Cultura de Células/métodos , Humanos , Modelos Biológicos , Organoides/citologia , Medicina Regenerativa , Reprodutibilidade dos Testes , Células-Tronco , Engenharia Tecidual/métodos
6.
Biomaterials ; 198: 135-145, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30174198

RESUMO

The intricate architecture of branched tissues and organs has fascinated scientists and engineers for centuries. Yet-despite their ubiquity-the biophysical and biochemical mechanisms by which tissues and organs undergo branching morphogenesis remain unclear. With the advent of three-dimensional (3D) culture models, an increasingly powerful and diverse set of tools are available for investigating the development and remodeling of branched tissues and organs. In this review, we discuss the application of 3D culture models for studying branching morphogenesis of the mammary gland and the mammalian lung in the context of normal development and disease. While current 3D culture models lack the cellular and molecular complexity observed in vivo, we emphasize how these models can be used to answer targeted questions about branching morphogenesis. We highlight the specific advantages and limitations of using 3D culture models to study the dynamics and mechanisms of branching in the mammary gland and mammalian lung. Finally, we discuss potential directions for future research and propose strategies for engineering the next generation of 3D culture models for studying tissue morphogenesis.

7.
J Allergy Clin Immunol ; 143(4): 1482-1495, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30170123

RESUMO

BACKGROUND: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. OBJECTIVES: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

8.
J Allergy Clin Immunol ; 143(1): 173-181.e10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30248356

RESUMO

BACKGROUND: Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. OBJECTIVE: We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. METHODS: Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. RESULTS: In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. CONCLUSIONS: Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Dermatite Atópica , Guanilato Ciclase , Queratinócitos , Mutação com Perda de Função , Proteínas de Membrana , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Adolescente , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Índice de Gravidade de Doença , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
9.
Curr Opin Biomed Eng ; 6: 66-73, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30345410

RESUMO

The architectural features of branching morphogenesis demonstrate exquisite reproducibility among various organs and species despite the unique functionality and biochemical differences of their microenvironment. The regulatory networks that drive branching morphogenesis employ cell-generated and passive mechanical forces, which integrate extracellular signals from the microenvironment into morphogenetic movements. Cell-generated forces function locally to remodel the extracellular matrix (ECM) and control interactions among neighboring cells. Passive mechanical forces are the product of in situ mechanical instabilities that trigger out-of-plane buckling and clefting deformations of adjacent tissues. Many of the molecular and physical signals that underlie buckling and clefting morphogenesis remain unclear and require new experimental strategies to be uncovered. Here, we highlight soft material systems that have been engineered to display programmable buckles and creases. Using synthetic materials to model physicochemical and spatiotemporal features of buckling and clefting morphogenesis might facilitate our understanding of the physical mechanisms that drive branching morphogenesis across different organs and species.

10.
Curr Biol ; 28(20): R1197-R1200, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30352189

RESUMO

Textbooks commonly describe epithelia as tissues composed of regular arrays of prism-shaped cells. A recent study combining mathematical modeling with quantitative imaging has uncovered the scutoid, a new shape that is necessary for epithelial cells to pack into curved tissues.

11.
Adv Exp Med Biol ; 1092: 57-67, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30368748

RESUMO

Cells communicate constantly with their surrounding extracellular matrix (ECM) to maintain homeostasis, using both mechanical and chemical signals. In cancer, abnormal signaling leads to stiffening of the ECM. A stiff microenvironment affects many aspects of the cell, including internal molecular signaling as well as behaviors such as motility and proliferation. Thus, cells and ECM interact in a feedback loop to drive matrix deposition and cross-linking, which alter the mechanical properties of the tissue. Stiffer tissue enhances the invasive potential of a tumor and decreases therapeutic efficacy. This chapter describes how specific molecular effects caused by an abnormally stiff tissue drive macroscopic changes that help determine disease outcome. A complete understanding may foster the generation of new cancer therapies.


Assuntos
Microambiente Celular , Matriz Extracelular , Neoplasias/patologia , Fenômenos Biomecânicos , Movimento Celular , Humanos , Transdução de Sinais
12.
J Cell Biol ; 217(10): 3319-3321, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194268

RESUMO

Smooth muscle-like cells can actively remodel epithelia, a mechanism common across developing tissues. In this issue, new work from Sirka et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201802144) demonstrates a novel mechanism for tumor suppression by smooth muscle-like myoepithelial cells of the mammary gland.


Assuntos
Células Epiteliais , Músculo Liso , Epitélio
14.
Artigo em Inglês | MEDLINE | ID: mdl-30249770

RESUMO

Smooth muscle is increasingly recognized as a key mechanical sculptor of epithelia during embryonic development. Smooth muscle is a mesenchymal tissue that surrounds the epithelia of organs including the gut, blood vessels, lungs, bladder, ureter, uterus, oviduct and epididymis. Smooth muscle is stiffer than its adjacent epithelium and often serves its morphogenetic function by physically constraining the growth of a proliferating epithelial layer. This constraint leads to mechanical instabilities and epithelial morphogenesis through buckling. Smooth muscle stiffness alone, without smooth muscle cell shortening, seems to be sufficient to drive epithelial morphogenesis. Fully understanding the development of organs that use smooth muscle stiffness as a driver of morphogenesis requires investigating how smooth muscle develops, a key aspect of which is distinguishing smooth muscle-like tissues from one another in vivo and in culture. This necessitates a comprehensive appreciation of the genetic, anatomical and functional markers that are used to distinguish the different subtypes of smooth muscle (for example, vascular versus visceral) from similar cell types (including myofibroblasts and myoepithelial cells). Here, we review how smooth muscle acts as a mechanical driver of morphogenesis and discuss ways of identifying smooth muscle, which is critical for understanding these morphogenetic events.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.


Assuntos
Células Epiteliais/fisiologia , Morfogênese/fisiologia , Músculo Liso/embriologia , Animais , Humanos
15.
Curr Opin Cell Biol ; 54: 98-105, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29890398

RESUMO

Morphogenesis encompasses the developmental processes that reorganize groups of cells into functional tissues and organs. The spatiotemporal patterning of individual cell behaviors is influenced by how cells perceive and respond to mechanical forces, and determines final tissue architecture. Here, we review recent work examining the physical mechanisms of tissue morphogenesis in vertebrate and invertebrate models, discuss how epithelial cells employ contractility to induce global changes that lead to tissue folding, and describe how tissue form itself regulates cell behavior. We then highlight novel tools to recapitulate these processes in engineered tissues.


Assuntos
Organogênese , Animais , Fenômenos Biomecânicos , Microambiente Celular , Células Epiteliais/citologia , Humanos , Modelos Biológicos , Morfogênese
17.
Front Cell Dev Biol ; 6: 21, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29560350

RESUMO

Cancer is a complex, heterogeneous group of diseases that can develop through many routes. Broad treatments such as chemotherapy destroy healthy cells in addition to cancerous ones, but more refined strategies that target specific pathways are usually only effective for a limited number of cancer types. This is largely due to the multitude of physiological variables that differ between cells and their surroundings. It is therefore important to understand how nature coordinates these variables into concerted regulation of growth at the tissue scale. The cellular microenvironment might then be manipulated to drive cells toward a desired outcome at the tissue level. One unexpected parameter, cellular membrane voltage (Vm), has been documented to exert control over cellular behavior both in culture and in vivo. Manipulating this fundamental cellular property influences a remarkable array of organism-wide patterning events, producing striking outcomes in both tumorigenesis as well as regeneration. These studies suggest that Vm is not only a key intrinsic cellular property, but also an integral part of the microenvironment that acts in both space and time to guide cellular behavior. As a result, there is considerable interest in manipulating Vm both to treat cancer as well as to regenerate organs damaged or deteriorated during aging. However, such manipulations have produced conflicting outcomes experimentally, which poses a substantial barrier to understanding the fundamentals of bioelectrical reprogramming. Here, we summarize these inconsistencies and discuss how the mechanical microenvironment may impact bioelectric regulation.

18.
J Cell Sci ; 131(8)2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29588395

RESUMO

The Wnt/ß-catenin pathway controls a variety of cellular behaviors, aberrant activation of which are associated with tumor progression in several types of cancer. The same cellular behaviors are also affected by the mechanical properties of the extracellular matrix (ECM) substratum, which induces signaling through integrins and integrin-linked kinase (ILK). Here, we examined the role of substratum stiffness in the regulation of cell proliferation downstream of Wnt3a. We found that treatment with Wnt3a increased proliferation of cells cultured on stiff substrata, with compliances characteristic of breast tumors, but not of cells on soft substrata, with compliances comparable to that of normal mammary tissue. Depleting ILK rendered cells unresponsive to Wnt3a on both substrata. Ectopic expression of ILK permitted Wnt3a to induce proliferation of cells on both microenvironments, although proliferation on soft substrata remained lower than that on stiff substrata. We further showed that ILK regulates expression of the Wnt receptor frizzled-1 (Fzd1), suggesting the presence of a positive feedback loop between Wnt3a, ILK and Fzd1. These findings suggest that tissue mechanics regulates the cellular response to Wnt under physiological and pathological microenvironmental conditions.This article has an associated First Person interview with the first author of the paper.


Assuntos
Matriz Extracelular/metabolismo , Receptores Frizzled/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Wnt3A/metabolismo , Proliferação de Células , Células Cultivadas , Humanos
19.
Cancer Res ; 78(9): 2277-2289, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29483094

RESUMO

Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFß, known inducers of epithelial-mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression.Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277-89. ©2018 AACR.


Assuntos
Transição Epitelial-Mesenquimal , Fatores de Transcrição da Família Snail/metabolismo , Microambiente Tumoral , Animais , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Instabilidade Genômica , Humanos , Metaloproteinase 3 da Matriz/metabolismo , Mecanotransdução Celular , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
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