Janton, Francis et al. (2017) International Worm Meeting "The DVA neuron promotes wakefulness via a cAMP-mediated mechanism."

Nelson, Matthew, Szurgot, Mary, Brown, Amelia, Sullivan, Nicole, Vance, Ryan, Janton, Francis

[International Worm Meeting, 2017]

Caenorhabditis elegans display unique forms of sleep: developmentally timed sleep (DTS) and stress-induced sleep (SIS), which are regulated by overlapping and distinct mechanisms (Trojanowski et al., 2015). The cyclic adenosine monophosphate (cAMP)/Protein Kinase A (PKA) pathway promotes arousal in invertebrates and vertebrates (Crocker and Sehgal, 2010). This signaling pathway is downregulated during DTS in C. elegans (Belfer et al., 2013) and we show the same is true for SIS. To determine where cAMP/PKA functions during sleep we have been expressing and activating the near infrared light-activated adenylyl cyclase called IlaC, which converts ATP into cAMP in the presence of red light, in candidate neurons (Ryu et al., 2014). We have chosen this tool because, unlike blue light, red light does not wake worms up during sleep. We find that pan-neuronal activation of IlaC reduces both DTS and SIS. Induction of cAMP in either the motor neurons, command interneurons or the sleep-regulating interneuron RIA is not sufficient to reduce DTS or SIS. However, activation of IlaC in the DVA interneuron, by expressing it from the full twk-16 promoter or using a DVA-specific enhancer from the twk-16 promoter (Puckett Robinson et al., 2013), significantly reduces SIS, DTS and non-physiological quiescence induced by neuropeptide over-expression (Nelson et al. 2013 and 2014). This suggests that DVA may be a common wake-promoting neuron downstream of distinct sleep promoting circuits. To confirm DVA's place in the known circuitry, we have expressed a cAMP biosensor, Epac1-camps (Shafer et al., 2008) in DVA and are currently measuring cAMP levels during sleep. What lies downstream of the cAMP/PKA pathway during DTS and SIS is still unclear. The cAMP response element binding protein (CREB) is a transcription factor known to play a role in C. elegans sleep and arousal, as well as arousal in mammals (Graves et al., 2003). CRH-1 is the C. elegans CREB homologue; and crh-1 loss-of-function mutants display increased amounts of DTS (Singh et al., 2014), which we have observed as well. We show evidence that CRH-1 functions downstream of cAMP/PKA in the nervous system during DTS, and are currently testing if DVA is the wake-promoting site of action. Surprisingly, we find that crh-1 mutants have reduced SIS, thus cAMP/PKA promotes arousal after SIS in DVA via distinct mechanisms.

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.

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.

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.

Juo P et al. (2007) Mol Biol Cell "CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans."

Kaplan JM, Harbaugh T, Garriga G, Juo P

[Mol Biol Cell, 2007]

Monitoring Editor: Francis Barr The proline-directed kinase CDK-5 plays a role in several aspects of neuronal development. Here we show that CDK-5 activity regulates the abundance of the glutamate receptor GLR-1 in the ventral cord of C. elegans and produces corresponding changes in GLR-1-dependent behaviors. Loss of CDK-5 activity results in decreased abundance of GLR-1 in the ventral cord, accompanied by accumulation of GLR-1 in neuronal cell bodies. Genetic analysis of cdk-5 and the clathrin adaptin unc-11 AP180 suggests that CDK-5 functions before endocytosis at the synapse. The scaffolding protein LIN-10/Mint-1 also regulates GLR-1 abundance in the nerve cord. CDK-5 phosphorylates LIN-10/Mint-1 in vitro and bidirectionally regulates the abundance of LIN-10/Mint-1 in the ventral cord. We propose that CDK-5 promotes the anterograde trafficking of GLR-1, and that phosphorylation of LIN-10 may play a role in this process.

Sri Koduri et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Role of Stargazin in Regulating Glutamate Gated Currents"

Sri Koduri, Andres Maricq, David Madsen, Craig Walker

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The majority of excitatory neurotransmitter function in vertebrates is mediated by ionotropic glutamate receptors (iGluRs). Reconstitution of iGluRs from C. elegans in Xenopus oocytes requires the auxiliary subunits SOL-1 and STG-1 (see abstracts by Francis et al., and Walker et al.). STG-1 shares weak sequence identity with vertebrate stargazin - a member of the family of glutamate receptor regulatory proteins known as TARPs (Transmembrane AMPA Receptor Regulatory Proteins). We have cloned stargazin homologues from Apis mellifera and Drosophila and co-expressed them with ionotropic glutamate receptor subunits in Xenopus oocytes. We found that the kinetics of the glutamate gated current were dependent upon the combination of the glutamate receptor subunit and the stargazin homologue. Apis STG, when co expressed with vertebrate GluR1, caused the channels to become non-desensitizing. We are currently analyzing chimeras made from Apis STG and vertebrate stargazin to determine which domain of the protein modifies the desensitization kinetics.

Nicholas Duclos et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identifying Genes Required for Signaling via the CAM-1 Receptor Tyrosine Kinase"

Nicholas Duclos, Andres Maricq, Michael Francis, Penelope Brockie

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Signaling at synapses requires that cells within the nervous system adopt the correct cell identity. Thus, both pre- and post-synaptic cells must express a specific compliment of molecules and establish the appropriate synaptic connections. We would like to understand how the C. elegans Ror receptor tyrosine kinase (RTK), CAM-1, contributes to these processes. Ror RTKs are highly expressed in the vertebrate nervous system as well as other tissues, yet the mechanism of signaling by these RTKs remains unclear. cam-1 encodes the only Ror RTK in C. elegans and is normally expressed in both the nervous system and in body wall muscles. In neurons, it has been associated with EGL-20, a Wnt signaling molecule, in regulating nervous system development (Forrester et al.). At the neuromuscular junction, it has been implicated in the stabilization and localization of the acetylcholine receptor subunit ACR-16 (Francis et al.). Overexpression of CAM-1 in either muscles or neurons produces dramatic overgrowth of cellular processes. In order to identify gene products that regulate Ror RTKs, we have initiated a forward screen for modifiers of CAM-1 function that takes advantage of this overexpression phenotype. Overexpression of CAM-1 in the command interneurons produces a dramatic change in cell morphology that is easily identifiable by the presence of numerous highly branched ectopic neuronal outgrowths. To identify genes that function in the same pathway as cam-1, we are screening for suppressors of this phenotype. We expect to find genes that encode upstream regulators of CAM-1 signaling, such as potential ligands, as well as downstream components of the CAM-1 signaling pathway. The identification of these genes will yield valuable insights in to the mechanisms of Ror RTK signaling. Francis MM, Evans SP, Jensen M, Madsen DM, Mancuso J, Norman KR, Maricq AV. The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction. Neuron 46: 581-594Forrester WC, Kim C, Garriga G. The C. elegans Ror RTK CAM-1 collaborates with EGL-20/Wnt to regulate cell migration. 168: 1951-1962

Clifford R et al. (1996) Midwest Worm Meeting "Searching for Proteins that Associate with GLD-1"

Clifford R, Giorgini F, Schedl TB

[Midwest Worm Meeting, 1996]

The gld-1 gene is involved in multiple aspects of C. elegans germline development. The primary, essential function of gld-1 is to direct oogenesis. In the absence of gld-1 activity, germ cells that would otherwise develop into oocytes enter meiosis normally but exit early pachytene to re-enter a mitotic cell cycle and form a tumor. The gene also plays nonessential roles in inhibiting premeiotic germ cell proliferation and promoting germ cells to adopt the male sexual fate [1,2]. gld-1 encodes a 463 amino acid protein that contains an evolutionarily conserved domain of ~200 amino acids that is also found in the mammalian Quaking and Sam68 proteins. A KH RNA binding motif lies within this domain [3]. Since GLD-1 appears to be localized exclusively to the cytoplasm, the protein is likely to regulate mRNA translation or stability [4]. To better understand how gld-1 acts in the control of germline development, we are using the yeast two-hybrid system [5] to isolate proteins that physically associate with the gene product. Through this approach we hope to identify regulators and cofactors of GLD-1. By screening an oligo(dT)-primed cDNA library (gift of R. Barstead) with a fragment of GLD-1 that includes the amino terminal half of the conserved domain, we identified the F28E10.1 gene product as a protein that specifically binds GLD-1. The F28E10.1 locus, which was identified by the C. elegans genome sequencing consortium, is predicted to encode a novel, hydrophilic protein of 118 kD. This gene maps to the region of chromosome IV that is deleted by mDf4 and may correspond to one of the lethal or sterile loci uncovered by the deficiency [6,7]. We are currently testing the biological relevance of the interaction between GLD-1 and F28E10.1 through in vivo assays. Progress will be reported. [1] Francis et al. 1995a. Genetics 139: 579-606; [2] Francis et al. 1995b. Genetics 139: 607-630; [3] Jones and Schedl. 1995. Genes Dev 9: 1491-1504; [4] Jones et al., in preparation; [5] Chien et al. 1991. PNAS 88: 9578-9582; [6] Rogalski and Riddle. 1988. Genetics 118: 61-74; [7] Cassada et al. 1981. Dev Biol 84: 193-205.