RESUMEN
We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.
Asunto(s)
Actinas/fisiología , Cadherinas/fisiología , Adhesión Celular/fisiología , Citoesqueleto/fisiología , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/fisiología , Actinas/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Separación Celular/métodos , Depsipéptidos/farmacología , Ratones , Tiazoles/farmacología , Tiazolidinas , Factores de TiempoRESUMEN
The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover, beta1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network organisation. Organotypic cultures of gut explants reveal that beta1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that beta1 integrins are required for the villi innervation in the small intestine. Our findings highlight the crucial roles played by beta1 integrins at various steps of enteric nervous system development.
Asunto(s)
Sistema Nervioso Entérico/embriología , Integrina beta1/genética , Cresta Neural/citología , Cresta Neural/embriología , Fenotipo , Animales , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/fisiología , Enfermedad de Hirschsprung/embriología , Inmunohistoquímica , Integrasas , Ratones , Modelos Genéticos , Mutación , Técnicas de Cultivo de Órganos , Proteínas ViralesRESUMEN
Using a dual pipette assay that measures the force required to separate adherent cell doublets, we have quantitatively compared intercellular adhesiveness mediated by Type I (E- or N-cadherin) or Type II (cadherin-7 or -11) cadherins. At similar cadherin expression levels, cells expressing Type I cadherins adhered much more rapidly and strongly than cells expressing Type II cadherins. Using chimeric cadherins, we found that the extracellular domain exerts by far the dominant effect on cell adhesivity, that of E-cadherin conferring high adhesivity, and that of cadherin-7 conferring low adhesivity. Type I cadherins were incorporated to a greater extent into detergent-insoluble cytoskeletal complexes, and their cytoplasmic tails were much more effective in disrupting strong adherent junctions, suggesting that Type II cadherins form less stable complexes with beta-catenin. The present study demonstrates compellingly, for the first time, that cadherins are dramatically different in their ability to promote intercellular adhesiveness, a finding that has profound implications for the regulation of tissue morphogenesis.
Asunto(s)
Proteínas Aviares/fisiología , Cadherinas/fisiología , Adhesión Celular , Animales , Proteínas Aviares/química , Proteínas Aviares/genética , Cadherinas/química , Cadherinas/genética , Línea Celular , Pollos , Uniones Intercelulares , Cinética , Microdominios de Membrana/fisiología , Transfección , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The ontogeny of neural crest cells (NCC) involves a number of orchestrated variety of derivatives, including components of the peripheral nervous system and melanocytes. Thus, it represents an excellent model system to investigate mechanisms controlling epithelial-mesenchymal transitions, cell migration and differentiation, as well as cell proliferation and death. We have established a new transgenic line expressing the Cre recombinase under the control of the human tissue plasminogen activator promoter (Ht-PA). The activity of the reporter in the Ht-PA-Cre/R26R embryos is observed as early as Theiler stage 12 in the cephalic mesenchyme. Later, the targeted cells include all the known derivatives of cranial, vagal, and trunk NCC, including craniofacial structures and cranial ganglia, cardiac and endocrine derivatives, melanocytes, peripheral, and enteric nervous system. At the vagal level, the location of presumptive enteric NCC differs from their avian counterparts in their ability to invade the mesenchyme lateral to the neural tube. In contrast to the Wnt1-Cre line, the Ht-PA-Cre line does not target the central nervous system and therefore renders it more specific for NCC. Our Ht-PA-Cre mice represent a novel model to specifically target conditional mutations in migratory NCC.
Asunto(s)
Integrasas/fisiología , Cresta Neural/citología , Regiones Promotoras Genéticas , Activador de Tejido Plasminógeno/genética , Proteínas Virales/fisiología , Animales , Movimiento Celular , Humanos , Ratones , Ratones Transgénicos , beta-Galactosidasa/genéticaRESUMEN
Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. We have generated mice with a neural crest cell-specific deletion of the beta1-integrin gene to analyse the role of beta1-integrins in neural crest cell migration and differentiation. This targeted mutation caused death within a month of birth. The loss of beta1-integrins from the embryo delayed the migration of Schwann cells along axons and induced multiple defects in spinal nerve arborisation and morphology. There was an almost complete absence of Schwann cells and sensory axon segregation and defective maturation in neuromuscular synaptogenesis. Thus, beta1-integrins are important for the control of embryonic and postnatal peripheral nervous system development.