RESUMEN
RNA tomography or tomo-seq combines mRNA sequencing and cryo-sectioning to spatially resolve gene expression. We have adapted this method for the nematode Caenorhabditis elegans to generate anteroposterior gene expression maps at near-cellular resolution. Here, we provide a detailed overview of the method and present two approaches: one that includes RNA isolation for maximum sensitivity and one that is suitable for partial automatization and is therefore less time-consuming. For complete details on the use and execution of this protocol, please refer to Ebbing et al. (2018).
Asunto(s)
Caenorhabditis elegans , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Tomografía/métodos , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/genéticaRESUMEN
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
Divergence of gene function and expression during development can give rise to phenotypic differences at the level of cells, tissues, organs, and ultimately whole organisms. To gain insights into the evolution of gene expression and novel genes at spatial resolution, we compared the spatially resolved transcriptomes of two distantly related nematodes, Caenorhabditis elegans and Pristionchus pacificus, that diverged 60-90 Ma. The spatial transcriptomes of adult worms show little evidence for strong conservation at the level of single genes. Instead, regional expression is largely driven by recent duplication and emergence of novel genes. Estimation of gene ages across anatomical structures revealed an enrichment of novel genes in sperm-related regions. This provides first evidence in nematodes for the "out of testis" hypothesis that has been previously postulated based on studies in Drosophila and mammals. "Out of testis" genes represent a mix of products of pervasive transcription as well as fast evolving members of ancient gene families. Strikingly, numerous novel genes have known functions during meiosis in Caenorhabditis elegans indicating that even universal processes such as meiosis may be targets of rapid evolution. Our study highlights the importance of novel genes in generating phenotypic diversity and explicitly characterizes gene origination in sperm-related regions. Furthermore, it proposes new functions for previously uncharacterized genes and establishes the spatial transcriptome of Pristionchus pacificus as a catalog for future studies on the evolution of gene expression and function.
Asunto(s)
Caenorhabditis elegans/genética , Evolución Molecular , Familia de Multigenes , Espermatozoides , Transcriptoma , Animales , Caenorhabditis elegans/metabolismo , Duplicación de Gen , Perfilación de la Expresión Génica , Genoma de los Helmintos , Masculino , Meiosis/genética , Filogenia , Espermatogénesis/genética , Testículo/fisiologíaRESUMEN
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.
RESUMEN
Directional migration of neurons and neuronal precursor cells is a central process in nervous system development. In the nematode Caenorhabditis elegans, the two Q neuroblasts polarize and migrate in opposite directions along the anteroposterior body axis. Several key regulators of Q cell polarization have been identified, including MIG-21, DPY-19/DPY19L1, the netrin receptor UNC-40/DCC, the Fat-like cadherin CDH-4 and CDH-3/Fat, which we describe in this study. How these different transmembrane proteins act together to direct Q neuroblast polarization and migration is still largely unknown. Here, we demonstrate that MIG-21 and DPY-19, CDH-3 and CDH-4, and UNC-40 define three distinct pathways that have partially redundant roles in protrusion formation, but also separate functions in regulating protrusion direction. Moreover, we show that the MIG-21, DPY-19 and Fat-like cadherin pathways control the localization and clustering of UNC-40 at the leading edge of the polarizing Q neuroblast, and that this is independent of the UNC-40 ligands UNC-6/netrin and MADD-4. Our results provide insight into a novel mechanism for ligand-independent localization of UNC-40 that directs the activity of UNC-40 along the anteroposterior axis.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Neuronas/citología , Neuronas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Movimiento Celular , Centrosoma/metabolismo , Ligandos , Transducción de SeñalRESUMEN
To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used spatial transcriptomics to generate high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, we have developed computational methods for discovering region- and tissue-specific genes. We have found extensive sex-specific gene expression differences in the germline and sperm and discovered genes that are specifically expressed in the male reproductive tract. These include a group of uncharacterized genes that encode small secreted proteins that are required for male fertility. We conclude that spatial gene expression maps provide a powerful resource for identifying tissue-specific gene functions in C. elegans. Importantly, we found that expression maps from different animals can be precisely aligned, enabling transcriptome-wide comparisons of gene expression patterns.