-
[
Wiley Interdiscip Rev Dev Biol,
2014]
Studies of the development of the Caenorhabditis elegans male have been carried out with the aim of understanding the basis of sexual dimorphism. Postembryonic development of the two C. elegans sexes differs extensively. Development along either the hermaphrodite or male pathway is specified initially by the X to autosome ratio. The regulatory events initiated by this ratio include a male-determining paracrine intercellular signal. Expression of this signal leads to different consequences in three regions of the body: the nongonadal soma, the somatic parts of the gonad, and the germ line. In the nongonadal soma, activity of the key Zn-finger transcription factor TRA-1 determines hermaphrodite development; in its absence, the male pathway is followed. Only a few genes directly regulated by TRA-1 are currently known, including members of the evolutionarily conserved, male-determining DM domain Zn-finger transcription factors. In the somatic parts of the gonad and germ line, absence of TRA-1 activity is not sufficient for full expression of the male pathway. Several additional transcription factors involved have been identified. In the germ line, regulatory genes for sperm development that act at the level of RNA in the cytoplasm play a prominent role.
-
[
Curr Opin Struct Biol,
2010]
RNA-binding proteins are critical effectors of gene expression. They guide mRNA localization, translation, and stability, and potentially play a role in regulating mRNA synthesis. The structural basis for RNA recognition by RNA-binding proteins is the key to understand how they target specific transcripts for regulation. Compared to other metazoans, nematode genomes contain a significant expansion in several RNA-binding protein families, including Pumilio-FBF (PUF), TTP-like zinc finger (TZF), and Argonaute-like (AGO) proteins. Genetic data suggest that individual members of each family have distinct functions, presumably due to sequence variations that alter RNA-binding specificity or protein interaction partners. In this review, we highlight example structures and identify the variable regions that likely contribute to functional divergence in nematodes.
-
[
Trends Cell Biol,
2019]
Fluorescent proteins have revolutionized biomedical research as they are easy to use for protein tagging, cope without fixation or permeabilization, and thus, enable live cell imaging in various models. Current methods allow easy and quick integration of fluorescent markers to endogenous genes of interest. In this review, we introduce the three central methods, zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and CRISPR, that have been widely used to manipulate cells or organisms. Focusing on CRISPR technology, we give an overview on homology-directed repair (HDR)-, microhomology-mediated end joining (MMEJ)-, and nonhomologous end joining (NHEJ)-based strategies for the knock-in of markers, figure out recent developments of the technique for highly efficient knock-in, and demonstrate pros and cons. We highlight the unique aspects of fluorescent protein knock-ins and pinpoint specific improvements and perspectives, like the combination of editing with stem cell derived organoid development.
-
[
Cell Biosci,
2016]
The generation of mutants and transgenes are indispensible for biomedical research. In the nematode Caenorhabditis elegans, a series of methods have been developed to introduce genome modifications, including random mutagenesis by chemical reagents, ionizing radiation and transposon insertion. In addition, foreign DNA can be integrated into the genome through microparticle bombardment approach or by irradiation of animals carrying microinjected extrachromosomal arrays. Recent research has revolutionized the genome engineering technologies by using customized DNA nucleases to manipulate particular genes and genomic sequences. Many streamlined editing strategies are developed to simplify the experimental procedure and minimize the cost. In this review, we will summarize the recent progress of the site-specific genome editing methods in C. elegans, including the Cre/LoxP, FLP/FRT, MosTIC system, zinc-finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease. Particularly, the recent studies of CRISPR/Cas9-mediated genome editing method in C. elegans will be emphatically discussed.
-
[
Biol Bull,
1998]
The identification of the molecular components of the developmental neurogenic programs in different organisms has revealed an astounding degree of conservation across phylogeny, suggesting that the basic mechanisms of neural development have also been conserved in evolution. One class of conserved neural regulatory genes, the LIM homeobox genes, encode transcription factors with two Zn-finger-like LIM domains and a DNA-binding homeodomain. Vertebrate members of this class have been implicated in neurogenesis by correlative expression evidence: e.g., the combinatorial expression of LIM homeobox genes in the vertebrate spinal cord suggested a "LIM-code" for specific motorneuronal targeting choices. Genetic analysis in Drosophila also demonstrated their essential role in axon pathfinding and the determination of neurotransmitter identity. The genome of the nematode Caenorhabditis elegans is almost completely sequenced, thus allowing the analysis of complete gene families in a metazoan organism. C. elegans contains seven LIM homeobox genes. Almost all C. elegans LIM homeobox genes fall into subclasses that are defined by the presence of similar genes from arthropods and vertebrates, suggesting a common origin for different subclasses of LIM homeobox genes.
-
[
Int J Mol Sci,
2016]
Caenorhabditis elegans, a 1 mm long free-living nematode, is a popular model animal that has been widely utilized for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle (development from egg to adult in 3.5 days at 20 C), well-annotated genome, simple morphology (comprising only 959 somatic cells in the hermaphrodite), and transparency (which facilitates non-invasive fluorescence observations). However, early approaches to introducing mutations in the C. elegans genome, such as chemical mutagenesis and imprecise excision of transposons, have required large-scale mutagenesis screens. To avoid this laborious and time-consuming procedure, genome editing technologies have been increasingly used in nematodes including C. briggsae and Pristionchus pacificus, thereby facilitating their genetic analyses. Here, I review the recent progress in genome editing technologies using zinc-finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in nematodes and offer perspectives on their use in the future.
-
[
ScientificWorldJournal,
2011]
Caenorhabditis elegans has a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream of C. elegans researchers is to establish a toolkit for the controlled manipulation of any loci within the genome. Although C. elegans is amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation.
-
[
Biochem Soc Trans,
2010]
We previously developed a transgenic Caenorhabditis elegans model of human tauopathy disorders by expressing human tau in nematode worm neurons to explore genetic pathways contributing to tau-induced neurodegeneration. This animal model recapitulates several hallmarks of human tauopathies, including altered behaviour, accumulation of detergent-insoluble phosphorylated tau protein and neurodegeneration. To identify genes required for tau neurotoxicity, we carried out a forward genetic screen for mutations that suppress tau neurotoxicity. We ultimately cloned the
sut-2 (suppressor of tau pathology-2) gene, mutations in which alleviate tau neurotoxicity in C. elegans. SUT-2 encodes a novel subtype of CCCH zinc-finger protein conserved across animal phyla. SUT-2 shares significant identity with the mammalian SUT-2 (MSUT-2). We identified components of the aggresome as binding partners of MSUT-2. Thus we hypothesize that MSUT-2 plays a role in the formation and/or clearance of protein aggregates. We are currently exploring the role of MSUT-2 in tauopathy using mammalian systems. The identification of
sut-2 as a gene required for tau neurotoxicity in C. elegans suggests new neuroprotective strategies targeting MSUT-2 that may be effective in modulating tau neurotoxicity in human tauopathy disorders.
-
[
Adv Exp Med Biol,
2011]
In normal development cell fusion is essential for organ formation and sexual reproduction. The nematode Caenorhabditis elegans has become an excellent system to study the mechanisms and developmental functions of cell-to-cell fusion. In this review we focus on the heterochronic regulation of cell fusion. Heterochronic genes control the timing of specific developmental events in C. elegans. The first microRNAs discovered were found as mutations that affect heterochronic development and cell-cell fusions. In addition numerous heterochronic transcription factors also control specific cell fusion events in C. elegans. We describe what is known about the heterochronic regulation of cell fusion of the epidermal seam cells. The fusogen AFF-1 was previously shown to mediate the fusion of the lateral epidermal seam cells. Here we provide evidence supporting the model in which LIN-29, the heterochronic Zinc-finger transcription factor that controls the terminal fusion of the seam cells, stimulates AFF-1 expression in the seam cells before they fuse. Therefore, the heterochronic gene LIN-29 controls AFF-1-mediated cell-cell fusion as part of the terminal differentiation program of the epidermal seam cells.
-
[
Vet Parasitol,
1999]
Nematode parasites of warm-blooded hosts use chemical and thermal signals in host-finding and in the subsequent resumption of development. The free-living nematode Caenorhabditis elegans is a useful model for investigating the chemo- and thermosensory neurons of such parasites, because the functions of its amphidial neurons are well known from laser microbeam ablation studies. The neurons found in the amphidial channel detect aqueous chemoattractants and repellants; the wing cells-flattened amphidial neurons-detect volatile odorants. The finger cells-digitiform amphidial neurons-are the primary thermoreceptors. Two neuron classes, named ADF and ASI, control entry into the environmentally resistant resting and dispersal dauer larval stage, while the paired ASJ neurons control exit from this stage. Skin-penetrating nematode parasites, i.e. the dog hookworm Ancylostoma caninum, and the threadworm, Strongyloides stercoralis, use thermal and chemical signals for host-finding, while the passively ingested sheep stomach worm, Haemonchus contortus, uses environmental signals to position itself for ingestion. Amphidial neurons presumably recognize these signals. In all species, resumption of development, on entering a host, is probably triggered by host signals also perceived by amphidial neurons. In the amphids of the A. caninum infective larva, there are wing- and finger-cell neurons, as well as neurons ending in cilia-like dendritic processes, some of which presumably recognize a sequence of signals that stimulate these larvae to attach to suitable hosts. The functions of these neurons can be postulated, based on the known functions of their homologs in C. elegans. The threadworm, S. stercoralis, has a complex life cycle. After leaving the host, soil-dwelling larvae may develop either to infective larvae (the life-stage equivalent of dauer larvae) or to free-living adults. As with the dauer larva of C. elegans, two neuron classes control this developmental switch. Amphidial neurons control chemotaxis to a skin extract, and a highly modified amphidial neuron, the lamellar cell, appears to be the primary thermoreceptor, in addition to having chemosensory function. The stomach worm, Haemonchus contortus, depends on ingestion by a grazing host. Once ingested, the infective larva is exposed to profound environmental changes in the rumen. These changes stimulate resumption of development in this species. We hypothesize that resumption of development is under the control of the ASJ neuronal pair. Identification of the neurons that control the infective process could provide the basis for entirely new approaches to parasite control involving interference with development at the time and place of initial host-contact.