Johnston RJ et al. (2010) Cell "Preview. A penetrating look at stochasticity in development."

Johnston RJ, Desplan C

[Cell, 2010]

In recent work published in Nature, Raj et al. (2010) use single mRNA molecule quantification to show that variation in gene expression in Caenorhabditis elegans increases in mutants displaying incomplete penetrance. They find that a bimodal response is triggered when noisy expression of an upstream regulator crosses a critical threshold.

Lee C et al. (2017) Bio Protoc "Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans."

Sorensen EB, Lee C, Seidel HS, Lynch TR, Crittenden SL, Kimble J

[Bio Protoc, 2017]

Single-molecule RNA fluorescence <i>in situ</i> hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj <i>et al.</i>, 2008 ; Lee <i>et al.</i>, 2016a ). We adapted this technique to visualize RNAs in the <i>C. elegans</i> whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee <i>et al.</i>, 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for <i>C. elegans</i> extruded gonads.

Schiffer, Jodie et al. (2021) International Worm Meeting "Caenorhabditis elegans processes sensory information to choose between freeloading and self-defense strategies"

McGowan, Natalie, Tam, Hannah, Stroustrup, Nicholas, Amrit, Francis Raj Ghandi, Heath, William, Stanley, Julian, Vogelaar, Abigail, Schiffer, Jodie, Servello, Francesco, Martin, Olivier, Xu, Yuyan, Stumbur, Stephanie, Apfeld, Javier, Eder, Matthias, Johnsen, Sean, Serkin, William, Brennan, Sarah, Ghazi, Arjumand

[International Worm Meeting, 2021]

Hydrogen peroxide is the preeminent chemical weapon that organisms use for combat. Individual cells rely on conserved defenses to prevent and repair peroxide-induced damage, but whether similar defenses might be coordinated across cells and tissues in animals remains poorly understood. Here, we screen a collection of sensory neuron genetic ablations in the nematode C. elegans to determine their effects on resistance to peroxide. We identify a neuronal circuit that processes information perceived by two of those sensory neurons to control the induction of hydrogen peroxide defenses in the organism. In the presence of E. coli, C. elegans'food source, the animal's neurons signal via TGFb-insulin/IGF1 relay to target tissues to repress expression of catalases and other hydrogen peroxide defenses. We found that catalases produced by E. coli can compensate for the animal's downregulation of catalases by depleting hydrogen peroxide from the local environment and thereby protecting C. elegans. This adaptive strategy is the first example of a multicellular organism modulating its defenses when it expects to freeload from the protection provided by molecularly orthologous defenses from another species.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

Broitman-Maduro G et al. (2011) Methods Cell Biol "In situ hybridization of embryos with antisense RNA probes."

Maduro MF, Broitman-Maduro G

[Methods Cell Biol, 2011]

Detection of transcripts in situ is a rapid means by which gene expression can be characterized in many systems. In the nematode, Caenorhabditis elegans, the ease with which transgenics can be made and the general reliability of reporter fusion expression patterns, have made this technique comparatively less popular than in other systems. There are, however, still applications in which in situ hybridization is desired, such as for maternally expressed genes, or in related species without established transgene methods. The most frequently used method of in situ hybridization uses DNA probes and formaldehyde fixation. A newer approach that permits single-transcript detection has been reported and will not be described here (Raj and Tyagi, 2010). Rather, we describe an alternative protocol that uses RNA probes with a different fixative. This approach has been applied to C. elegans and related nematodes, providing reliable, sensitive detection of endogenous transcripts.

J Rogers et al. (1999) International C. elegans Meeting "Feeding For Phenotypes"

J Hubbard, J Rogers

[International C. elegans Meeting, 1999]

We were intrigued by the recent results of Timmons and Fire (1998) suggesting that RNAi-induced phenotypes could be observed by simply feeding worms with bacteria that are producing the dsRNA of interest. Given the potency of RNAi in the germ line, we wondered if this technique could be used to produce germline-defective phenotypes of interest to our lab. In addition, we sought to apply the technique to identify genes involved in germline function that may otherwise cause a loss-of-function embryonic lethal phenotype. To test this possibility, we collected several cDNAs from genes that produce loss-of-function germline-defective phenotypes (some of which also cause embryonic or larval lethatity) and introduced them into the "double T7" vector described by Timmons and Fire. We are testing these constructs by feeding synchronous populations of L1 larvae with bacteria that express dsRNA and scoring them in the adult for germline defects. Preliminary data suggests that germline phenotypes can be obtained in this manner, but that they may be restricted to events that occur relatively late in germline development. For example, gld-1 dsRNA fed to L1 larvae produced an abnormal oocyte phenotype characteristic of partial loss-of-function alleles of gld-1 (class E and F; Francis et al., 1995) at 60% penetrance. Progeny of unaffected adults were then grown on RNAi-producing bacteria. In these animals we observed the more severe tumorous phenotype in which progression through meiotic prophase is altered and oogenesis does not occur (Francis et al., 1995).

Frederic Horndli et al. (2008) C.elegans Neuronal Development Meeting "Identification of ACR-16 Specific Trafficking and Targeting Components"

Andres Maricq, Frederic Horndli, Michael Jensen

[C.elegans Neuronal Development Meeting, 2008]

Three types of receptors are present at the neuromuscular junction in C. elegans: the levamisole acetylcholine receptor (AChR) complex LevR (UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8), the nicotinic AChR ACR-16 and the GABA receptor UNC-49. Both ACR-16 and the LevR complex respond to cholinergic inputs, with ACR-16 contributing to 70% of the EPSP (Francis M.M.., et al., 2005). Although a mechanism for stabilization and clustering of ACR-16 has been described (Francis MM., et al., 2005), very little is known, about how AChR receptors are transported and inserted at the NMJ. Using RNAi targeting of known transport motors and cargo adaptor proteins, in combination with either unc-29 (x29) or acr-16 (ok789) deletion backgrounds we have successfully identified the kinesin-1 transport complex (composed of unc-116, klc-2, unc-16 and unc-14 (Byrd DT., et aI., 2001, Sakamoto R., et al., 2004)) as an ACR-16 specific transport complex. Indeed, RNAi and deletion mutants for all elements of this complex in combination with unc-29 (x29), exhibit almost complete paralysis as well as ACR-16 GFP mislocalization. In addition, the same RNAi and deletion mutants for the elements of the kinesin-1 complex do not affect acr-16(ok789) movement nor UNC-29 GFP and UNC-49 GFP localization. Encouraged by these results we have started a whole genome RNAi screening approach using the unc-29(x29) and acr-16(ok789) background to discover more elements related to ACR-16 and UNC-29 trafficking, targeting, insertion and stabilization.

Tetsunari Fukushige et al. (2001) Worm Breeder's Gazette "Monoclonal Antibody MH33 Recognizes a Gut-specific Intermediate Filament"

Tetsunari Fukushige, Jim McGhee

[Worm Breeder's Gazette, 2001]

The monoclonal antibody MH33 was produced by Francis and Waterston (1985) and detects a structure beneath the microvilli of intestinal cells, probably the terminal web. At the comma stage of embryogenesis, staining is both apical and cytoplasmic; apical staining becomes predominant in later stages up to the adult. The MH33 staining pattern differs from that produced by MH27, which recognizes a component of the adherens junction at the basolateral side of gut cells. To identify the MH33 antigen, we screened a C. elegans expression library (kindly provided by Dr. B. Barstead) by using MH33 antibody (kindly provided by Drs. Hresko and Waterson) and isolated 24 positive clone from ~ 100,000 plaques screened. A cross hybridization experiment indicated that these positive clones all encoded the same product. Several of the largest inserts were sequenced and identified gene F10C1.7, which encodes an intermediate filament. Dodement et al., (1994) had previously identified this protein as intermediate filament b2. Two observations suggest that we have indeed cloned the correct gene. (1) Westerns could easily detect a ~60kD band in extracts from twenty worms carrying a complete transgene; (Francis and Waterston (1991) originally reported that MH33 identifies bands at 62 and 64 kD, and Dodement et al. showed that alternative splicing gives rise to two forms of b2 that differ in size by ~ 2kD). (2) A five kb fragment from the F10C1.7 5!-flanking region, fused to pPD96.04, drives gut specific expression, starting at comma stage and continuing in all later stages. The MH33-detected gut-specific intermediate filament gene should be useful as a marker of early gut development (the gene is a potential direct target of elt-2, for example, and contains the usual suspiciously located WGATAR sites) and as a probe of intestinal cell structure, in particular the generation of apical-basal polarity during gut development.

Kim, Dong hyun et al. (2011) International Worm Meeting "Dampening of expression oscillations by co-regulation of a microRNA and its target."

van Oudenaarden, Alexander, Kim, Dong Hyun

[International Worm Meeting, 2011]

Failure to properly execute temporal developmental programs in a multi-cellular organism can result in severe morphological defects and often sterility. In nematode C. elegans, the timing of larval stage specific developmental events are tightly controlled by "heterochronic genes" which form dosage temporal gradients during development. This gradient is mainly achieved by negative regulation via short non-coding regulatory RNAs, or microRNA(miRNA)s. During early post-embryonic stages, lin-4 miRNA mediates the down-regulation of LIN-14 which is required for timely execution of a series of developmental events. A number of reports have demonstrated the dosage dependent activities of these genes. More specifically, LIN-14 is believed to exhibit a temporally decreasing dosage gradient, running from high to low, triggering different stage-specific programs during early larval growth. We have quantitatively measured the transcript levels of lin-14 and the promoter activity of lin-4 miRNA in staged C. elegans larvae using single-molecule RNA in situ hybridization method (Raj et al., 2008). Surprisingly, we found both lin-14 and its negative post-transcriptional regulator lin-4 to exhibit an in-phase oscillatory transcription activity which peaks every intermolt throughout the animal body. In wild-type animals, this co-regulation leads to an efficient dampening of the lin-14 transcript level oscillation and results in a step-wise decreasing lin-14 mRNA temporal gradient. We have also confirmed this at the individual cell level, including lateral seam cells and tail hypodermis. We have evidence that the underlying oscillation is not larval stage specific and is neither due to increased gene copy number in dividing cells nor regulatory feedback between lin-14 and/or lin-4. Thus, we speculate the source of oscillation is linked to organismal growth. Taking these observations into account, we speculate lin-4 miRNA and its target lin-14 form an incoherent feed forward loop (IFFL) modulating oscillatory input signal into discrete graded output and propose a novel functional role of miRNA-mediated IFFL as a developmental clock. Reference - Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, and Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877-879 (2008).

de Chadarevian S (1998) Studies of History & Philosophy of Science "Of Worms and programmes - Caenorhabditis elegans and the study of development."

de Chadarevian S

[Studies of History & Philosophy of Science, 1998]

In 1963, just a year after the researchers of the Medical Research Council (MRC) Unit of Molecular Biology in Cambridge, joined by some other research groups, has moved from various scattered and makeshift buildings in the courtyard of the Physics Department to a lavishly funded four-storey laboratory, B. Lush, the Principal Medical Officer of the MRC, came to inquire about their plans for future expansion. He indicated that the MRC wished to build the laboratory up to what the principal researchers considered its 'final size' until their retirement, which meant planning ahead for at least 15 years. This surprising move was doubtless prompted by the recent award of the Nobel Prize to three members of the laboratory, Max Perutz, John Kendrew and Francis Crick, for their work on the molecular structure of proteins and nucleic acids. The triple award had propelled the new Laboratory of Molecular Biology into the limelight, and the MRC was interested in securing optimal research conditions for this prestigious group of researchers.