Establishment of a tissue-specific RNAi system in C. elegans

Qadota, Hiroshi; Inoue, Minako; Hikita, Takao; Köppen, Mathias; Hardin, Jeffrey D.; Amano, Mutsuki; Moerman, Donald G.; Kaibuchi, Kozo

doi:10.1016/j.gene.2007.06.020

Cited by 196 publications

(177 citation statements)

References 39 publications

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“…The egl-8 gene is not known to be expressed in the epidermis (21). As expected, feeding egl-30 or egl-8 dsRNA to the NR222 strain, which restricts RNAi to the epidermis (25), had no effect on pathogen sensitivity, life span, or aldicarb resistance (Table S2). These results are consistent with the predominant role of egl-30 and egl-8 in the intestine for immune function and in the neurons for life span regulation.…”

Section: Loss Of Gqα and Plcβ Functions Conferred Enhanced Daf-16-regsupporting

confidence: 65%

Systemic and cell intrinsic roles of Gqα signaling in the regulation of innate immunity, oxidative stress, and longevity in Caenorhabditis elegans

Kawli

Wu²,

Tan³

2010

Proc. Natl. Acad. Sci. U.S.A.

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Signal transduction pathways that regulate longevity, immunity, and stress resistance can profoundly affect organismal survival. We show that a signaling module formed by the G protein alpha subunit, Gqα, and one of its downstream signal transducer phospholipase C β (PLCβ) can differentially affect these processes. Loss of Gqα and PLCβ functions result in increased sensitivity to pathogens and oxidative stress but confer life span extension. Gqα and PLCβ modulate life span and immunity noncell autonomously by affecting the activity of insulin/IGF1 signaling (IIS). In addition, Gqα and PLCβ function cell autonomously within the intestine to affect the activity of the p38 MAPK pathway, an important component of Caenorhabditis elegans immune and oxidative stress response. p38 MAPK activity in the intestine is regulated by diacylglycerol levels, a product of PLCβ's hydrolytic activity. We provide genetic evidence that life span is largely determined by IIS, whereas p38 MAPK signaling is the primary regulator of oxidative stress in PLCβ mutants. Pathogen sensitivity of Gqα and PLCβ mutants is a summation of the beneficial effects of decreased IIS through reduced neuronal secretion and the detrimental effects of reduced activity of intestinal p38 MAPK. We propose a model whereby Gqα signaling differentially regulates pathogen sensitivity, oxidative stress, and longevity through cell autonomous and noncell autonomous effects on p38 MAPK and insulin/IGF1 signaling, respectively.insulin/IGF-1 signaling | p38 MAPK signaling | infection | aging A ppropriate responses to endogenous and exogenous threats at the organismal level are achieved by the coordination and integration of both cell-intrinsic and systemic signaling events. The conserved insulin/IGF1 signaling (IIS) pathway regulates life span, stress responses, and immunity. In Caenorhabditis elegans, activation of the insulin/IGF1-like receptor (IGFR) DAF-2 results in phosphorylation and retention of the forkhead transcription factor DAF-16 in the cytoplasm (1). Reduction in IGFR function results in increased life span and oxidative stress resistance in C. elegans (2, 3), Drosophila (4), and mice (5). Oxidative stress response is also regulated by the p38 MAPK cascade, which in C. elegans comprises of a three-tiered kinase cascade leading to phosphorylation by a MAPK kinase homolog SEK-1 and activation of the p38 MAPK homolog PMK-1 (6). Loss of either sek-1 or pmk-1 function results in increased sensitivity to oxidative stress (7). The p38 MAPK pathway affects oxidative stress resistance cell autonomously in part through regulation of the Nrf family transcription factor SKN-1 in the intestine (8, 9).IIS and p38 MAPK signaling contribute to C. elegans immunity. Diminished IIS confers pathogen resistance because of derepression of DAF-16 transcriptional activity and increased immuneeffector gene expression (10, 11), whereas diminished p38 MAPK signaling confers increased pathogen sensitivity (12); these pathways regulate distinct sets of immune-related genes (13-15). Re...

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Section: Loss Of Gqα and Plcβ Functions Conferred Enhanced Daf-16-regsupporting

confidence: 65%

Systemic and cell intrinsic roles of Gqα signaling in the regulation of innate immunity, oxidative stress, and longevity in Caenorhabditis elegans

Kawli

Wu²,

Tan³

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Bacaj and Shaham (2007) describe a useful method to circumvent this limitation by using cell-specific rescue of heat-shock factor-1 (hsp-1) to restrict heat-shock-induced expression of a gene of interest to cells that express active hsf-1 (Bacaj and Shaham 2007). A similar strategy can be applied to rescue of genes required for RNAi, such as rde-1 (Tabara et al 1999) to restrict RNAi to certain times and/or places (Qadota et al 2007). While these strategies are useful, expression of the rescuing gene (e.g., hsf-1) is dependent upon continued expression from the promoter driving it and/or its activity (e.g., binding and activating the heat-shock promoter).…”

Section: Discussionmentioning

confidence: 99%

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

Voutev

Hubbard

2008

Genetics

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We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.T WO-COMPONENT gene expression systems are indispensable tools to probe molecular mechanisms underlying development. Because control can be exerted by each component independently, exquisite temporal and spatial control of gene activation or repression can be achieved. Using different promoter combinations to drive each component of these systems, additional control can be obtained beyond that afforded by heat-inducible or tissue-specific promoters alone. Site-specific recombination systems such as the FLP/FRT system have been used to control gene expression by ''FLP-out'': a recombinase-catalyzed intramolecular excision of spacer DNA that lies between tandemly oriented FRT sites. The spacer includes a transcriptional stop so that prior to activation of the FLP recombinase (and subsequent FLP-out) the gene downstream of the spacer is not transcribed (Golic and Lindquist 1989;Struhl and Basler 1993; Figure 1A). After the FRT-containing cassette is excised by the FLP recombinase, the downstream gene is brought into proximity to the promoter and is expressed (reporter 2 in Figure 1A). This system and related systems have proven quite powerful and flexible in model organisms including Drosophila and mouse (see Branda and Dymecki 2004, for review;McGuire et al. 2004). However, prior to our study presented here, and a recently published study (Davis et al. 2008), these systems had not been developed for use in Caenorhabditis elegans.An ideal FLP-out system provides the means to generate both loss-and gain-of-function effects in a spatially and temporally controlled manner. In addition, wild-type gene expression can be turned on in particular cells at particular times in an otherwise mutant background. In organisms where transgenes can be reliably inserted in single copy, FLP-out can also be used to eliminate wild-type gene expression by excision of an FRT-flanked wild-type cassette in a mutant background. In C. elegans, the most common methods for generating transgenic C. elegans introduce multiple copies of transgenes on extrachromosomal arrays (Stinchcomb et al. 1985;Mello et al. 1991;Kelly et al. 1997). ...

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“…Mutant rde-1 worms are resistant to RNAi, but restoring tissue-specific expression of the wild-type rde-1 cDNA in these mutants confers RNAi sensitivity to a specific tissue (27). We depleted cua-1 in wildtype N2, WM27 (rde-1 mutant, RNAi insensitive), VP303 (P nhx-2 ::RDE-1, intestine only RNAi), WM118 (P myo-3 ::RDE-1, muscle only RNAi), and NR222 (P lin-26 ::RDE-1, hypodermis only RNAi) worm strains (13).…”

Section: Cua-1 Expression Is Regulated By Dietarymentioning

confidence: 99%