RESUMEN
Members of the Wnt family of secreted glycoproteins regulate cell migration through distinct canonical and noncanonical signaling pathways. Studies of vertebrate development and disease have shown that these pathways can have opposing effects on cell migration, but the mechanism of this functional interplay is not known. In the nematode Caenorhabditis elegans, a switch from noncanonical to canonical Wnt signaling terminates the long-range migration of the QR neuroblast descendants, providing a tractable system to study this mechanism in vivo. Here, we show that noncanonical Wnt signaling acts through PIX-1/RhoGEF, while canonical signaling directly activates the Slt-Robo pathway component EVA-1/EVA1C and the Rho GTPase-activating protein RGA-9b/ARHGAP, which are required for migration inhibition. Our results support a model in which cross-talk between noncanonical and canonical Wnt signaling occurs through antagonistic regulation of the Rho GTPases that drive cell migration.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Movimiento Celular , Proteínas Activadoras de GTPasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Receptores Inmunológicos/metabolismo , Vía de Señalización Wnt , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Movimiento Celular/genética , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Receptores Inmunológicos/genética , Proteínas RoundaboutRESUMEN
Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans, combined with an extensive genetic toolbox and the ease of growing large tightly synchronized populations makes it an exceptional model organism for the application of such approaches. However, the difficulty to dissociate and isolate single cells from larval stages has been a major constraint to this kind of studies. Here, we describe an improved protocol for dissociation and preparation of single cell suspensions from developmentally synchronized populations of C. elegans L1 larvae. Our protocol has been empirically optimized to allow efficient FACS-based purification of large number of single cells from rare cell types, for subsequent extraction and sequencing of their mRNA.