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1.
Cell ; 173(3): 762-775.e16, 2018 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-29677517

RESUMEN

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.


Asunto(s)
Mecanotransducción Celular , Interferencia de ARN , Receptores Acoplados a Proteínas G/fisiología , Animales , Materiales Biocompatibles , Calcio/metabolismo , Línea Celular Tumoral , Células Endoteliales/fisiología , Endotelio Vascular/citología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Concentración de Iones de Hidrógeno , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Resistencia al Corte , Estrés Mecánico , Resistencia Vascular
2.
Nature ; 541(7636): 176-181, 2017 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-28002412

RESUMEN

Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2+ vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired Hering-Breuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults.


Asunto(s)
Apnea/fisiopatología , Canales Iónicos/metabolismo , Pulmón/fisiología , Pulmón/fisiopatología , Mecanotransducción Celular/fisiología , Reflejo/fisiología , Animales , Animales Recién Nacidos , Apnea/genética , Muerte , Femenino , Canales Iónicos/deficiencia , Canales Iónicos/genética , Masculino , Mecanotransducción Celular/genética , Ratones , Ganglio Nudoso/metabolismo , Reflejo/genética , Respiración , Células Receptoras Sensoriales/metabolismo , Volumen de Ventilación Pulmonar
3.
Dev Biol ; 468(1-2): 101-109, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-32979334

RESUMEN

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.


Asunto(s)
Apoptosis , Diencéfalo/embriología , Mesencéfalo/embriología , Tubo Neural/embriología , Animales , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Ratones , Ratones Transgénicos , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo
4.
Proc Natl Acad Sci U S A ; 115(50): 12817-12822, 2018 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-30482854

RESUMEN

PIEZO1 is a cation channel that is activated by mechanical forces such as fluid shear stress or membrane stretch. PIEZO1 loss-of-function mutations in patients are associated with congenital lymphedema with pleural effusion. However, the mechanistic link between PIEZO1 function and the development or function of the lymphatic system is currently unknown. Here, we analyzed two mouse lines lacking PIEZO1 in endothelial cells (via Tie2Cre or Lyve1Cre) and found that they exhibited pleural effusion and died postnatally. Strikingly, the number of lymphatic valves was dramatically reduced in these mice. Lymphatic valves are essential for ensuring proper circulation of lymph. Mechanical forces have been implicated in the development of lymphatic vasculature and valve formation, but the identity of mechanosensors involved is unknown. Expression of FOXC2 and NFATc1, transcription factors known to be required for lymphatic valve development, appeared normal in Tie2Cre;Piezo1cKO mice. However, the process of protrusion in the valve leaflets, which is associated with collective cell migration, actin polymerization, and remodeling of cell-cell junctions, was impaired in Tie2Cre;Piezo1cKO mice. Consistent with these genetic findings, activation of PIEZO1 by Yoda1 in cultured lymphatic endothelial cells induced active remodeling of actomyosin and VE-cadherin+ cell-cell adhesion sites. Our analysis provides evidence that mechanically activated ion channel PIEZO1 is a key regulator of lymphatic valve formation.


Asunto(s)
Canales Iónicos/metabolismo , Linfangiogénesis/fisiología , Sistema Linfático/metabolismo , Sistema Linfático/fisiología , Vasos Linfáticos/metabolismo , Vasos Linfáticos/fisiología , Actomiosina/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Factores de Transcripción Forkhead/metabolismo , Uniones Intercelulares/metabolismo , Uniones Intercelulares/fisiología , Transporte Iónico/fisiología , Ratones , Factores de Transcripción NFATC/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo
5.
Life Sci Alliance ; 6(2)2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36446523

RESUMEN

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.


Asunto(s)
Canales Iónicos , Regeneración , Células Satélite del Músculo Esquelético , Animales , Ratones , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Canales Iónicos/genética , Canales Iónicos/fisiología , Músculo Esquelético/fisiología , Mioblastos/fisiología , Transducción de Señal , Células Satélite del Músculo Esquelético/fisiología , Regeneración/genética , Regeneración/fisiología
6.
Sci Rep ; 12(1): 4197, 2022 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-35273307

RESUMEN

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.


Asunto(s)
Canales Iónicos , Células Mesangiales , Renina , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Aparato Yuxtaglomerular/metabolismo , Riñón/metabolismo , Células Mesangiales/metabolismo , Ratones , Renina/genética , Renina/metabolismo
7.
Genes Cells ; 15(5): 501-12, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20384787

RESUMEN

In utero electroporation is widely used to study neuronal development and function by introducing plasmid DNA into neural progenitors during embryogenesis. This is an effective and convenient method of introducing plasmid DNA into neural precursors and is suitable for manipulating gene expression in cells of the CNS. However, the applicability of this technique is comparatively limited to neuronal research, as the plasmid DNA introduced into neural progenitors during embryogenesis is diluted by cell proliferation and is not stably maintained in glial cells generated around and after birth. To overcome this limitation, we applied the Tol2 transposon system, which integrates a transgene into the genome of the host cell, to in utero electroporation. With this system, we confirmed that the transgene was effectively maintained in the progeny of embryonic neural precursors, astrocytes and oligodendrocytes. Using the glial promoters GFAP and S100beta, targeted and stable expressions of transgenes in glia were obtained, which enabled the expression of different transgenes simultaneously in neurons and glia. Glia-targeted expression of the transgene that causes neuronal migration defect was achieved without the defect. Thus, use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia-neuron interactions in vivo.


Asunto(s)
Elementos Transponibles de ADN , Electroporación/métodos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Transferencia de Gen , Neuroglía/fisiología , Neuronas/fisiología , Transgenes , Animales , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/citología , Neuronas/citología , Embarazo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/genética , Proteínas S100/metabolismo , Células Madre/citología , Células Madre/fisiología , Transposasas/metabolismo
9.
Dev Cell ; 27(6): 621-34, 2013 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-24369835

RESUMEN

Apoptotic cells are observed in the early developing brain. Apoptosis deficiency is proposed to cause brain overgrowth, but here we show that brain malformations in apoptosis-deficient mutants are due to insufficient brain ventricle expansion as a result of uncompleted cranial neural tube closure. Apoptosis eliminates Fgf8-expressing cells in the anterior neural ridge (ANR), which acts as an organizing center of the forebrain by producing FGF8 morphogen. Deficiency of apoptosis leads to the accumulation of undead and nonproliferative cells in the ventral part of the ANR. The undead cells in apoptosis-deficient mutants express Fgf8 continuously, which perturbs gene expression in the ventral forebrain. Thus, apoptosis within a specific subdomain of the ANR is required for correct temporal elimination of an FGF8-producing region within a limited developmental time window, thereby ensuring proper forebrain development.


Asunto(s)
Apoptosis , Factor Apoptótico 1 Activador de Proteasas/fisiología , Encéfalo/patología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Neuronas/patología , Animales , Encéfalo/metabolismo , Ciclo Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Hibridación Fluorescente in Situ , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Transducción de Señal
10.
J Cell Biol ; 195(6): 1047-60, 2011 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-22162136

RESUMEN

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)-based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.


Asunto(s)
Apoptosis , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Confocal/métodos , Tubo Neural/embriología , Neurulación , Imagen de Lapso de Tiempo/métodos , Animales , Caspasas/metabolismo , Femenino , Elementos Aisladores , Masculino , Ratones , Ratones Transgénicos , Morfogénesis , Tubo Neural/metabolismo , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo
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