ABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cell Movement , GTPase-Activating Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neural Stem Cells/metabolism , Receptors, Immunologic/metabolism , Wnt Signaling Pathway , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Cell Movement/genetics , Gene Expression Regulation , Nerve Tissue Proteins/genetics , Neural Stem Cells/cytology , Receptors, Immunologic/genetics , Roundabout ProteinsABSTRACT
High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of Caenorhabditis elegans research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hands-on time and sustainability compared to current standardized DNA extraction protocols, while equally effective. This work indicates that it is useful to compare methods from different model systems, such as mammalian organoids and invertebrate nematodes, to find useful alternatives for research methodologies.
ABSTRACT
Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.
Subject(s)
Calcium , Heart , Myocytes, Cardiac , Regeneration , Sarcomeres , Zebrafish , Animals , Calcium/physiology , Cell Proliferation , Heart/physiology , Myocytes, Cardiac/physiology , Sarcomeres/physiology , Zebrafish/physiologyABSTRACT
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).