A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans.
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ABSTRACT:
SUBMITTER: Wurmthaler LA
PROVIDER: S-EPMC6353947 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin-inducible degron (AID) system to immobilize both adult and larval hermaphrodite and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.
| S-EPMC8527506 | biostudies-literature
Project description:Developmental programs are executed by tightly controlled gene regulatory pathways. Here, we combined the unique sample retrieval capacity afforded by laser capture microscopy with analysis of mRNA abundance by CEL-Seq (cell expression by linear amplification and sequencing) to generate a spatiotemporal gene expression map of the Caenorhabditis elegans syncytial germline from adult hermaphrodites and males. We found that over 6000 genes exhibit spatiotemporally dynamic expression patterns throughout the hermaphrodite germline, with two dominant groups of genes exhibiting reciprocal shifts in expression at late pachytene during meiotic prophase I. We found a strong correlation between restricted spatiotemporal expression and known developmental and cellular processes, indicating that these gene expression changes may be an important driver of germ cell progression. Analysis of the male gonad revealed a shift in gene expression at early pachytene and upregulation of subsets of genes following the meiotic divisions, specifically in early and late spermatids, mostly transcribed from the X chromosome. We observed that while the X chromosome is silenced throughout the first half of the gonad, some genes escape this control and are highly expressed throughout the germline. Although we found a strong correlation between the expression of genes corresponding to CSR-1-interacting 22G-RNAs during germ cell progression, we also found that a large fraction of genes may bypass the need for CSR-1-mediated germline licensing. Taken together, these findings suggest the existence of mechanisms that enable a shift in gene expression during prophase I to promote germ cell progression.
| S-EPMC6216576 | biostudies-literature
Project description:Ribozymes, RNA molecules that catalyze the cleavage of RNA substrates, provide an interesting alternative to the RNA interference (RNAi) approach to gene inactivation, especially given the fact that RNAi seems to trigger an immunological response. Unfortunately, the limited substrate specificity of ribozymes is considered to be a significant hurdle in their development as molecular tools. Here, we report the molecular engineering of a ribozyme possessing a new biosensor module that switches the cleavage activity from 'off' (a 'safety lock') to 'on' solely in the presence of the appropriate RNA target substrate. Both proof-of-concept and the mechanism of action of this man-made riboswitch are demonstrated using hepatitis delta virus ribozymes that cleave RNA transcripts derived from the hepatitis B and C viruses. To our knowledge, this is the first report of a ribozyme bearing a target-dependent module that is activated by its RNA substrate, an arrangement which greatly diminishes non-specific effects. This new approach provides a highly specific and improved tool with significant potential for application in the fields of both functional genomics and gene therapy.
| S-EPMC549572 | biostudies-literature
Project description:Development of the Caenorhabditis elegans vulva serves as a paradigm for intercellular signaling during animal development. In wild-type animals, the somatic gonadal anchor cell generates the LIN-3/EGF ligand to induce vulval fates in the underlying hypodermis, whereas FBF, FOG-1, and FOG-3 control germ-line development. Here we report that FBF functions redundantly with FOG-1 and FOG-3 to control vulval induction: animals lacking FBF and either FOG-1 or FOG-3 have multiple vulvae, the Muv phenotype. The fog; fbf Muv phenotype is generated by aberrant induction of vulval precursor cells (VPCs): in wild-type animals, three VPCs are induced to form a single vulva, but, in fog; fbf mutants, four or five VPCs are typically induced, resulting in ectopic vulvae. Laser ablation experiments and mosaic analyses demonstrate that the germ line is critical for the fog; fbf Muv phenotype. Consistent with that site of action, we detect FBF and FOG-1 in the germ line but not in the VPCs. The simplest interpretation is that FOG-1, FOG-3, and FBF act in the germ line to influence vulval fates. The LIN-3/EGF ligand may be the germ-line signal to the VPCs: the fog; fbf Muv phenotype depends on LIN-3 activity, and the lin-3 3' UTR possesses an FBF binding element. Our findings reveal new insights into germ line-to-soma signals and the role of PUF proteins in animal development.
| S-EPMC1334682 | biostudies-literature
Project description:We describe a method for conditional regulation of gene expression based on the processing of an intron cassette. The RNA processing factor MEC-8 is necessary for the function of the Caenorhabditis elegans touch receptor neurons; mec-8 mutants are touch insensitive. We show here that this insensitivity involves the loss of MEC-8-dependent splicing of mec-2, which encodes a component of the mechanosensory transduction complex. MEC-8 is needed to remove the ninth intron in mec-2 pre-mRNA to form the longest of three mRNAs, mec-2a. Without MEC-8, splicing causes the termination of the transcript. Inclusion of mec-2 intron 9 is sufficient to convey mec-8-dependent regulation on other genes and, in mec-8(u218ts) mutants, resulted in their temperature-dependent expression. Because mec-8 is expressed ubiquitously in embryos and extensively in larvae, this system should produce temperature-sensitive expression for most genes. As an example, we report a strain that exhibits temperature-dependent RNA interference.
| S-EPMC2862115 | biostudies-literature
Project description:Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO2 sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO2 causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO2. Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO2-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed.
| S-EPMC5282578 | biostudies-literature
Project description:BackgroundThe ascarosides form a family of small molecules that have been isolated from cultures of the nematode Caenorhabditis elegans. They are often referred to as "dauer pheromones" because most of them induce formation of long-lived and highly stress resistant dauer larvae. More recent studies have shown that ascarosides serve additional functions as social signals and mating pheromones. Thus, ascarosides have multiple functions. Until now, it has been generally assumed that ascarosides are constitutively expressed during nematode development.Methodology/principal findingsCultures of C. elegans were developmentally synchronized on controlled diets. Ascarosides released into the media, as well as stored internally, were quantified by LC/MS. We found that ascaroside biosynthesis and release were strongly dependent on developmental stage and diet. The male attracting pheromone was verified to be a blend of at least four ascarosides, and peak production of the two most potent mating pheromone components, ascr#3 and asc#8 immediately preceded or coincided with the temporal window for mating. The concentration of ascr#2 increased under starvation conditions and peaked during dauer formation, strongly supporting ascr#2 as the main population density signal (dauer pheromone). After dauer formation, ascaroside production largely ceased and dauer larvae did not release any ascarosides. These findings show that both total ascaroside production and the relative proportions of individual ascarosides strongly correlate with these compounds' stage-specific biological functions.Conclusions/significanceAscaroside expression changes with development and environmental conditions. This is consistent with multiple functions of these signaling molecules. Knowledge of such differential regulation will make it possible to associate ascaroside production to gene expression profiles (transcript, protein or enzyme activity) and help to determine genetic pathways that control ascaroside biosynthesis. In conjunction with findings from previous studies, our results show that the pheromone system of C. elegans mimics that of insects in many ways, suggesting that pheromone signaling in C. elegans may exhibit functional homology also at the sensory level. In addition, our results provide a strong foundation for future behavioral modeling studies.
| S-EPMC3058051 | biostudies-literature
Project description:Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.
| S-EPMC4256423 | biostudies-literature
Project description:L1 ligase (L1L) molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5'-to-3' phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA world hypothesis. L1L crystal structure captures two distinct conformations that differ by a reorientation of one of the stems by around 80Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a three-state/two-step process. The first step involves a large-amplitude swing that reorients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network.
| S-EPMC3509952 | biostudies-literature
Project description:One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A) G-protein-coupled receptor (GPCR) chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH) and two nociceptive phasmid sensory neurons (PHA, PHB). We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of cases, the dauer-specific alterations of GPCR reporter expression in specific neuron classes are maintained during postdauer life and in some case new patterns are induced post-dauer, demonstrating that GPCR gene expression may serve as traits of life history. Taken together, our resource provides an entry point for functional studies and also offers a host of molecular markers for studying molecular patterning and plasticity of the nervous system.
| S-EPMC5749674 | biostudies-literature