cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans

The GAL4–UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4–UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit

C. elegans rrf-1 Mutations Maintain RNAi Efficiency in the Soma in Addition to the Germline

Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene sod-3; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools

A Mechanistic Basis for the Coordinated Regulation of Pharyngeal Morphogenesis in Caenorhabditis elegans by LIN-35/Rb and UBC-18–ARI-1

Genetic redundancy, whereby two genes carry out seemingly overlapping functions, may in large part be attributable to the intricacy and robustness of genetic networks that control many developmental processes. We have previously described a complex set of genetic interactions underlying foregut development in the nematode Caenorhabditis elegans. Specifically, LIN-35/Rb, a tumor suppressor ortholog, in conjunction with UBC-18–ARI-1, a conserved E2/E3 complex, and PHA-1, a novel protein, coordinately regulates an early step of pharyngeal morphogenesis involving cellular re-orientation. Functional redundancy is indicated by the observation that lin-35; ubc-18 double mutants, as well as certain allelic combinations of pha-1 with either lin-35 or ubc-18, display defects in pharyngeal development, whereas single mutants do not. Using a combination of genetic and molecular analyses, we show that sup-35, a strong recessive suppressor of pha-1–associated lethality, also reverts the synthetic lethality of lin-35; ubc-18, lin-35; pha-1, and ubc-18 pha-1 double mutants. SUP-35, which contains C2H2-type Zn-finger domains as well as a conserved RMD-like motif, showed a dynamic pattern of subcellular localization during embryogenesis. We find that mutations in sup-35 specifically suppress hypomorphic alleles of pha-1 and that SUP-35, acting genetically upstream of SUP-36 and SUP-37, negatively regulates pha-1 transcription. We further demonstrate that LIN-35, a transcriptional repressor, and UBC-18–ARI-1, a complex involved in ubiquitin-mediated proteolysis, negatively regulate SUP-35 abundance through distinct mechanisms. We also show that HCF-1, a C. elegans homolog of host cell factor 1, functionally antagonizes LIN-35 in the regulation of sup-35. Our cumulative findings piece together the components of a novel regulatory network that includes LIN-35/Rb, which functions to control organ morphogenesis. Our results also shed light on general mechanisms that may underlie developmental genetic redundancies as well as principles that may govern complex disease traits

Nucleolar Proteins Suppress Caenorhabditis elegans Innate Immunity by Inhibiting p53/CEP-1

The tumor suppressor p53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, and cell cycle regulation. A genetic screen for negative regulators of innate immunity in Caenorhabditis elegans led to the identification of a mutation in NOL-6, a nucleolar RNA-associated protein (NRAP), which is involved in ribosome biogenesis and conserved across eukaryotic organisms. Mutation or silencing of NOL-6 and other nucleolar proteins results in an enhanced resistance to bacterial infections. A full-genome microarray analysis on animals with altered immune function due to mutation in nol-6 shows increased transcriptional levels of genes regulated by a p53 homologue, CEP-1. Further studies indicate that the activation of innate immunity by inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1. Since nucleoli and p53/CEP-1 are conserved, our results reveal an ancient immune mechanism by which the nucleolus may regulate immune responses against bacterial pathogens

Pulsed Irradiation Improves Target Selectivity of Infrared Laser-Evoked Gene Operator for Single-Cell Gene Induction in the Nematode C. elegans

Methods for turning on/off gene expression at the experimenter’s discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application. Here, using the nematode Caenorhabditis elegans (C. elegans) as a subject, we have made improvements in IR-LEGO. For better spatial control of heating, a pulsed irradiation method using an optical chopper was introduced. As a result, single cells of C. elegans embryos as early as the 2-cell stage and single neurons in ganglia can be induced to express genes selectively. In addition, the introduction of site-specific recombination systems to IR-LEGO enables the induction of gene expression controlled by constitutive and cell type-specific promoters. The strategies adopted here will be useful for future applications of IR-LEGO to other organisms