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[
Methods Mol Biol,
2020]
This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes
kri-1 and
ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.
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[
WormBook,
2008]
The role of neuropeptides in modulating behavior is slowly being elucidated. With the sequencing of the C. elegans genome, the extent of the neuropeptide genes in C. elegans can be determined. To date, 113 neuropeptide genes encoding over 250 distinct neuropeptides have been identified. Of these, 40 genes encode insulin-like peptides, 31 genes encode FMRFamide-related peptides, and 42 genes encode non-insulin, non-FMRFamide-related neuropeptides. As in other systems, C. elegans neuropeptides are derived from precursor molecules that must be post-translationally processed to yield the active peptides. These precursor molecules contain a single peptide, multiple copies of a single peptide, multiple distinct peptides, or any combination thereof. The neuropeptide genes are expressed extensively throughout the nervous system, including in sensory, motor, and interneurons. In addition, some of the genes are also expressed in non-neuronal tissues, such as the somatic gonad, intestine, and vulval hypodermis. To address the effects of neuropeptides on C. elegans behavior, animals in which the different neuropeptide genes are inactivated or overexpressed are being isolated. In a complementary approach the receptors to which the neuropeptides bind are also being identified and examined. Among the knockout animals analyzed thus far, defects in locomotion, dauer formation, egg laying, ethanol response, and social behavior have been reported. These data suggest that neuropeptides have a modulatory role in many, if not all, behaviors in C. elegans.
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[
WormBook,
2006]
Most rapid excitatory synaptic signaling is mediated by glutamatergic neurotransmission. An important challenge in neurobiology is to understand the molecular architecture of functional glutamatergic synapses. By combining the techniques of genetics, molecular biology and electrophysiology in C. elegans we have the potential to identify and characterize the molecules that contribute to the function of glutamatergic synapses. In C. elegans both excitatory and inhibitory ionotropic glutamate receptors are linked to neural circuits and behavior. Genetic analysis has identified genes required for receptor expression, trafficking, localization, stabilization and function at synapses. Significantly, novel proteins required for glutamate receptor function have been discovered in the worm. These advances may also lead to a better understanding of glutamatergic signaling in vertebrates.
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Embryos are unique in their combination of pluripotency, three-dimensionality, and the swiftness of subcellular and developmental rearrangements. In some species, it is possible to observe the entire organism taking form within a microscope field. Capturing the spatial and temporal dynamic complexity of embryogenesis tests the limits of both culture and microscopy techniques. Observing specific fluorescently labeled components during embryonic development promises to reveal the roles of organelles and molecules in a native and reproducible context. However, to gain a thorough understanding of such dynamic biological systems, one must record events of interest as they occur, while limiting the perturbations caused by the observation techniques. In this chapter, we discuss our experiences using the relatively new technology of multiphoton laser scanning microscopy to examine the development of mammalian and nematode embryos in four dimensions.
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[
1993]
In the past 10 years, the remarkable discovery has been made that molecular mechanisms of development are conserved among all animals, and that many of the same molecular components appear in signal transduction pathways of all eukaryotes from yeast to man. This mechanistic conservation means that molecules can be studied in the organism in which their properties are most transparent; general principles or specific predictions made from work in one organism can subsequently be explored in other organisms. This chapter reviews aspects of nervous system development that have been studied using genetic approaches in two simple invertebrates, the fruit fly Drosophila melanogaster (herein referred to as Drosophila) and the soil nematode worm Caenorhabditis elegans (C. elegans). The nervous systems of both of these organisms are extremely simple compared to those of mammals....
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[
2000]
At fertilization, the calm of oogenesis ends and the egg abruptly begins a flurry of activity. Many crucial steps - decisions concerning when and where to divide, specification of cell fates, and establishment of body axes - rely on materials the egg contains at that moment. In many animals, the first few hours of life proceed with little or no transcription. As a result, developmental regulation at these early stages is dependent on maternal cytoplasm rather than the zygotic nucleus. The regulatory molecules accumulated during oogenesis might, in principle, be of any type, including RNA and protein. It is clear that mRNAs present in the egg before fertilization - so-called maternal mRNAs - play a particularly prominent role in early decisions. Viewed from this perspective, it is not surprising that oocytes and early embryos display an impressive array of posttranscriptional regulatory mechanisms, controlling mRNA stability, localization, and translation.
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[
WormBook,
2016]
In C. elegans, mutants that are defective in muscle function and/or structure are easy to detect and analyze since: 1) body wall muscle is essential for locomotion, and 2) muscle structure can be assessed by multiple methods including polarized light, electron microscopy (EM), Green Fluorescent Protein (GFP) tagged proteins, and immunofluorescence microscopy. The overall structure of the sarcomere, the fundamental unit of contraction, is conserved from C. elegans to man, and the molecules involved in sarcomere assembly, maintenance, and regulation of muscle contraction are also largely conserved. This review reports the latest findings on the following topics: the transcriptional network that regulates muscle differentiation, identification/function/dynamics of muscle attachment site proteins, regulation of the assembly and maintenance of the sarcomere by chaperones and proteases, the role of muscle-specific giant protein kinases in sarcomere assembly, and the regulation of contractile activity, and new insights into the functions of the dystrophin glycoprotein complex.
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[
1996]
At fertilization, the calm of oogenesis is broken, and the egg abruptly begins a flurry of activity. Many crucial steps - decisions concerning when and where to divide, specification of cell fates, and establishment of body axes - rely on materials the egg contains at that moment. In many animals, the first few hours of life proceed with little or no transcription. As a result, developmental regulation at these early stages is dependent on maternal cytoplasm, rather than the zygotic nucleus. The regulatory molecules accumulated during oogenesis might, in principle, be of any type, including RNA and protein. It is now clear that messenger RNAs present in the egg before fertilization (so-called maternal mRNAs) have a prominent role in early decisions. Viewed from this perspective, it is not surprising that oocytes and early embryos display an impressive array of posttrancriptional regulatory mechanisms, controlling mRNA stability, localization, and translation.
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[
1978]
A small sightless worm crawling among particles of soil and decaying vegetation must have a variety of chemical senses to locate bacteria for food and to avoid poisons and predators. What chemicals are sensed? How many different kinds of receptor molecules are there? On which neurons are the receptors located? How sensitive are these neurons? How is the detection of a chemical communicated to the worm's central nervous system and converted into a behavioral response? All of these questions have been addressed in studies of the soil nematode Caenorhabditis elegans. This organism has recently become the subject of intensive genetic, behavioral and anatomical studies. The behavior that has been examined in most detail is chemotaxis. This chapter will review what is known about C. elegans chemotaxis and will present a number of new observations. The results will be interpreted in terms of a specific model of chemoreceptor function. The problem of analysis of central nervous system processing of chemosensory neuron information will be discussed briefly.
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[
Cellular and Molecular Biology,
1989]
Myosin is assembled into thick filaments of distinct lengths and substructures in phylogenetically and histologically diverse muscles. In these different muscles, specific proteins are associated with myosin in the assembled filaments. In the nematode Caenorhabditis elegans, the major protein component is paramyosin which assembles with two myosin isoforms about a separate core structure. At least six non-myosin proteins are associated with the core structures. Previous models of myosin assembly have emphasized a linear sequence of steps in which myosin molecules themselves are involved in nucleation, elongation and termination of individual filaments. Nematode muscle mutants accumulate assemblages of multiple thich filaments which also appear at low levels in wild-type. The effects of various alterations of myosin myosin levels upon the assembly of the two myosins and the existence of these multi-filament assemblages suggest a possible alternative model for myosin assembly. In this model, a cycle in which multiple thick filaments nucleate from a common structure is driven by synthesis of a