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1.
Methods Mol Biol ; 2206: 39-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754809

RESUMO

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

2.
J Cell Sci ; 133(5)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31444283

RESUMO

Implanting biomaterials in tissues leads to inflammation and a foreign body response (FBR), which can result in rejection. Here, we live image the FBR triggered by surgical suture implantation in a translucent zebrafish model and compare with an acute wound response. We observe inflammation extending from the suture margins, correlating with subsequent avascular and fibrotic encapsulation zones: sutures that induce more inflammation result in increased zones of avascularity and fibrosis. Moreover, we capture macrophages as they fuse to become multinucleate foreign body giant cells (FBGCs) adjacent to the most pro-inflammatory sutures. Genetic and pharmacological dampening of the inflammatory response minimises the FBR (including FBGC generation) and normalises the status of the tissue surrounding these sutures. This model of FBR in adult zebrafish allows us to live image the process and to modulate it in ways that may lead us towards new strategies to ameliorate and circumvent FBR in humans.This article has an associated First Person interview with the first author of the paper.

3.
PeerJ ; 7: e6445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30918748

RESUMO

Green roofs, which are roofs with growing substrate and vegetation, can provide habitat for arthropods in cities. Maintaining a diversity of arthropods in an urban environment can enhance the functions they fill, such as pest control and soil development. Theory suggests that the creation of a heterogeneous environment on green roofs would enhance arthropod diversity. Several studies have examined how arthropod diversity can be enhanced on green roofs, and particularly whether substrate properties affect the arthropod community, but a gap remains in identifying the effect of substrate heterogeneity within a green roof on the arthropod community. In this paper, it is hypothesized that creating heterogeneity in the substrate would directly affect the diversity and abundance of some arthropod taxa, and indirectly increase arthropod diversity through increased plant diversity. These hypotheses were tested using green roof plots in four treatments of substrate heterogeneity: (1) homogeneous dispersion; (2) mineral heterogeneity-with increased tuff concentration in subplots; (3) organic heterogeneity-with decreased compost concentrations in subplots; (4) both mineral and organic heterogeneity. Each of the four treatments was replicated twice on each of three roofs (six replicates per treatment) in a Mediterranean region. There was no effect of substrate heterogeneity on arthropod diversity, abundance, or community composition, but there were differences in arthropod communities among roofs. This suggests that the location of a green roof, which can differ in local climatic conditions, can have a strong effect on the composition of the arthropod community. Thus, arthropod diversity may be promoted by building green roofs in a variety of locations throughout a city, even if the roof construction is similar on all roofs.

4.
EMBO Mol Med ; 10(11)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30143543

RESUMO

Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC-based proteomics to identify the orphan G protein-coupled receptor GPRC5A as a novel hypoxia-induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti-apoptotic target gene BCL2L1 Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF-GPRC5A-YAP axis as a critical mediator of the hypoxia-induced adaptive response and a potential target for cancer therapy.


Assuntos
Adaptação Fisiológica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/patologia , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Adaptação Fisiológica/efeitos dos fármacos , Animais , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Transcrição Genética/efeitos dos fármacos , Peixe-Zebra
5.
EMBO J ; 37(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29866703

RESUMO

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.


Assuntos
Macrófagos/fisiologia , Neovascularização Fisiológica , Cicatrização/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Diagnóstico por Imagem , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Peixe-Zebra/genética
6.
Cell Stem Cell ; 21(1): 107-119.e6, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28686860

RESUMO

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.


Assuntos
Linhagem da Célula/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Proteínas de Homeodomínio/metabolismo , Mioblastos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Proteínas de Homeodomínio/genética , Camundongos , Mioblastos/citologia , Proteínas de Peixe-Zebra/genética
7.
Science ; 353(6295): aad9969, 2016 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-27198673

RESUMO

Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.


Assuntos
Divisão Celular/fisiologia , Rastreamento de Células/métodos , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Animais Geneticamente Modificados , Divisão Celular/genética , Células Clonais , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/lesões , Mutação , Fator Regulador Miogênico 5/genética , Miogenina/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Transgenes , Peixe-Zebra
8.
Results Probl Cell Differ ; 56: 49-76, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25344666

RESUMO

Current evidence indicates that post-embryonic muscle growth and regeneration in amniotes is mediated almost entirely by stem cells derived from muscle progenitor cells (MPCs), known as satellite cells. Exhaustion and impairment of satellite cell activity is involved in the severe muscle loss associated with degenerative muscle diseases such as Muscular Dystrophies and is the main cause of age-associated muscle wasting. Understanding the molecular and cellular basis of satellite cell function in muscle generation and regeneration (myogenesis) is critical to the broader goal of developing treatments that may ameliorate such conditions. Considerable knowledge exists regarding the embryonic stages of amniote myogenesis. Much less is known about how post-embryonic amniote myogenesis proceeds, how adult myogenesis relates to embryonic myogenesis on a cellular or genetic level. Of the studies focusing on post-embryonic amniote myogenesis, most are post-mortem and in vitro analyses, precluding the understanding of cellular behaviours and genetic mechanisms in an undisturbed in vivo setting. Zebrafish are optically clear throughout much of their post-embryonic development, facilitating their use in live imaging of cellular processes. Zebrafish also possess a compartment of MPCs, which appear similar to satellite cells and persist throughout the post-embryonic development of the fish, permitting their use in examining the contribution of these cells to muscle tissue growth and regeneration.


Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Animais , Padronização Corporal/genética , Humanos , Músculo Esquelético/embriologia , Músculo Esquelético/patologia , Doenças Musculares/genética , Doenças Musculares/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Regeneração , Células-Tronco/citologia , Peixe-Zebra/embriologia
9.
Nature ; 512(7514): 314-8, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-25119043

RESUMO

Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.


Assuntos
Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Somitos/citologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Biomarcadores/análise , Movimento Celular , Quimiocina CXCL12/análise , Quimiocina CXCL12/metabolismo , Embrião de Galinha , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Músculos/citologia , Músculos/metabolismo , Mutação/genética , Somitos/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas Wnt/análise , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/genética
10.
FEBS J ; 280(17): 4074-88, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23607511

RESUMO

The cellular basis for mammalian muscle regeneration has been an area of intense investigation over recent decades. The consensus is that a specialized self-renewing stem cell, termed the satellite cell, plays a major role during the process of regeneration in amniotes. How broadly this mechanism is deployed within the vertebrate phylogeny remains an open question. A lack of information on the role of cells analogous to the satellite cell in other vertebrate systems is even more unexpected given the fact that satellite cells were first designated in frogs. An intriguing aspect of this debate is that a number of amphibia and many fish species exhibit epimorphic regenerative processes in specific tissues, whereby regeneration occurs by the dedifferentiation of the damaged tissue, without deploying specialized stem cell populations analogous to satellite cells. Hence, it is feasible that a cellular process completely distinct from that deployed during mammalian muscle regeneration could operate in species capable of epimorphic regeneration. In this minireview, we examine the evidence for the broad phylogenetic distribution of satellite cells. We conclude that, in the vertebrates examined so far, epimorphosis does not appear to be deployed during muscle regeneration, and that analogous cells expressing similar marker genes to satellite cells appear to be deployed during the regenerative process. However, the functional definition of these cells as self-renewing muscle stem cells remains a final hurdle to the definition of the satellite cell as a generic vertebrate cell type.


Assuntos
Desenvolvimento Muscular/fisiologia , Mioblastos/citologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia , Peixe-Zebra/fisiologia , Animais , Mioblastos/fisiologia , Células Satélites de Músculo Esquelético/fisiologia
11.
PLoS Biol ; 9(10): e1001168, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21990962

RESUMO

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.


Assuntos
Nadadeiras de Animais/crescimento & desenvolvimento , Evolução Biológica , Peixes/crescimento & desenvolvimento , Desenvolvimento Muscular , Pelve/crescimento & desenvolvimento , Nadadeiras de Animais/anatomia & histologia , Animais , Animais Geneticamente Modificados , Peixes/genética , Pelve/anatomia & histologia , Filogenia , Somitos/transplante , Especificidade da Espécie
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