Bennett, Heather L et al. (2011) International Worm Meeting "A role for heterochronic genes in regulating C.elegans quiescence."

Corkins, Mark E, Anderson, Edward, Singh, Komudi, Hart, Anne C, Bennett, Heather L

[International Worm Meeting, 2011]

Sleep is an essential biological process that is regulated by circadian rhythms, homeostatic regulation and arousal threshold. The nematode Caenorhabitdis elegans undergoes a sleep-like state called quiescence that accompanies each larval molt (Raizen et al., 2008). Many pathways regulate larval development, including heterochronic genes and the Notch signaling pathway. Heterochronic genes control timing of cell differentiation and molting of larval cuticle. In Drosophila, circadian rhythm proteins cycle to regulate sleep behavior. In C.elegans, circadian rhythm homologs in the heterochronic pathway regulate the fusion of hypodermal seam cells during late development (Jeon et al., 1999, Banerjee et al., 2005). Recently, a temporal role in C. elegans molting was suggested for the Notch signaling pathway. Gain of function alleles of a Notch receptor gene, lin-12, result in precocious cell fate changes (Solomon et al., 2007). We have found that Notch also plays a role in the regulation of quiescence. Increased or decreased Notch signaling results in increased quiescence and altered arousal thresholds during the last larval stage (L4) to adult (A) transition (Singh et al., in press). However, the connection between circadian rhythm homologs, heterochronic genes, and Notch in the regulation of quiescence remains unclear. We are assessing gain and loss of function alleles of heterochronic genes acting in the L4/A transition to determine if they play a role in regulating quiescence. Demonstrating a genetic interaction between heterochronic genes, circadian rhythm proteins, and the Notch signaling pathway will be the first step in delineating mechanisms that coordinate cell fate and behavioral changes during development.

Bennett, Heather L. et al. (2013) International Worm Meeting "Notch DSL ligand lag-2 is required for C. elegans lethargus quiescence."

Bennett, Heather L., Hart, Anne C., Singh, Komudi, Huang, Huiyan

[International Worm Meeting, 2013]

Sleep is an evolutionarily conserved behavior defined as a rapidly reversible period of inactivity with decreased sensory responsiveness. Despite this knowledge, the genetic underpinnings, mechanisms of sleep regulation, and the specific tissues involved in this behavior remain elusive. C. elegans undergoes sleep-like quiescence during lethargus in coordination with each larval cuticle molt (Raizen et al., 2008). Our laboratory has shown that Notch signaling regulates quiescence, as perturbations in the Notch pathway change the arousal thresholds and amount of quiescence during the last larval lethargus during transition from L4 stage to adult (Singh et al., 2011). We find that decreased function of lag-2, which encodes a Notch DSL ligand, results in increased quiescence and reduced arousal thresholds during the L4 to adult lethargus. However, it remains unclear how specific changes in lag-2 gene function disrupt aspects of this behavior. We are now characterizing the role of lag-2 in regulating behavioral quiescence. The specific cells where LAG-2 functions in this behavior and when lag-2 is required to regulate quiescence is being determined. The knowledge gained from addressing these aims will help us understand the role of Notch signaling, particularly the role of lag-2 in regulating C. elegans quiescence and may illuminate evolutionary conserved mechanisms required to regulate sleep across species.

Huang, Huiyan et al. (2015) International Worm Meeting "NCA sodium leak channels and gap junctions regulate sleep and arousal in C. elegans."

Huang, Huiyan, Zhu, Chen-Tseh, Bennett, Heather, Skuja, Lukas, Hart, Anne, Hayden, Dustin

[International Worm Meeting, 2015]

During sleep, animals exhibit a heightened arousal threshold and decreased behavioral response to external stimuli. The molecular and cellular mechanisms underlying these changes are not fully understood. We report here that the activity of the C. elegans NALCN sodium leak channel, NCA, is critical for normal sleep and arousal. Sodium leak channels regulate resting membrane potential, thus contributing to the overall neuronal excitability (PMID:17448995, PMID:23522043). We found that diminished NCA activity in C. elegans robustly increased arousal threshold during sleep bouts in L4-to-adult lethargus, consistent with decreased excitability. Quantity of sleep was only modestly increased in these animals, whereas heightened NCA activity dramatically decreased sleep. Loss of cGMP-dependent protein kinase EGL-4 suppressed NCA loss of function defects. This suggests a model in which increased arousal threshold during sleep requires down-regulation of the sodium leak channel, which, directly or indirectly, activates the EGL-4 protein kinase. Others have suggested that Drosophila sodium leak channel drives the pigment dispersing factor (PDF) neuropeptide release, regulating daily rhythmic behavior (PMID:16364900, PMID:20362452). However, in C. elegans, losing PDF-1, one of the two PDF neuropeptides, had no impact on animals with increased NCA function. Intriguingly, loss of the PDF receptor increased arousal threshold, reminiscent of decreased NCA function.NCA channels, cooperatively with gap junctions, are required for normal response to general anesthetics (PMID:3576211) and function in the premotor interneurons to regulate neuronal excitability (PMID:25716181, PMID: 21489288). Consistent with these findings, loss of UNC-7 or UNC-9 gap junction proteins partially suppressed the increased sleep of animals with diminished NCA function. Moreover, loss of UNC-7 in premotor interneurons or loss of UNC-9 in motor neurons significantly decreased sleep and lowered arousal threshold during sleep. Conversely, overexpressing UNC-7 and UNC-9 in adult animals was sufficient to induce anachronistic sleep. Combined, these results emphasize the conservation and importance of neuronal activity during sleep, demonstrate a role for gap junctions in sleep, and identify possible targets for the modulation of sleep entry and maintenance.

Singh, Komudi et al. (2011) International Worm Meeting "Notch signaling regulates many aspects of quiescence during C. elegans lethargus."

Singh, Komudi, Bennett, Heather L., Sorkac, Altar, Dilorio, Michael A., Hart, Anne C.

[International Worm Meeting, 2011]

Sleep/sleep-like behavior is universal across the animal kingdom. Yet, the molecular mechanisms underlying this behavior are not well understood. During the molt between each larval stage, C. elegans shed their cuticle and exhibit behavioral quiescence. Work from the Raizen laboratory has shown that quiescence shares many characteristics with sleep including diminished activity, rapidly reversibility, and decreased responsiveness to sensory stimuli. We examine here that the impact of Notch signaling on C. elegans quiescence. osm-11 encodes one of the five DOS family proteins that, with C. elegans DSL family ligands like LAG-2, activate LIN-12 and GLP-1 Notch receptors. Over-expression (OE) of osm-11 in adults induced anachronistic quiescence that was similar to molting quiescence and genetic epistasis studies suggested that osm-11(OE) mediated adult quiescence acts via Notch receptors and via pathways known to regulate C. elegans molting quiescence. To determine if the Notch pathway regulates larval molting quiescence, we developed a simple assay system using a microfluidic chip designed by Shawn Lockery to measure quiescence during L4-to-adult (L4/A) lethargus. Analysis of gain of function, loss of function, and transgenic animals revealed that altered Notch signaling impacts multiple elements of L4/A quiescence including amount of quiescence, arousal threshold, basal activity, and duration of lethargus. Our on-going studies suggest that C. elegans quiescence is a complex behavior composed of multiple elements and that Notch receptors may act in various sites to independently regulate these behavioral components of quiescence. For example, GLP-1 Notch receptor functions in sensory neurons to regulate arousal, but may act elsewhere to regulate basal activity. Notch signaling mediated regulation of quiescence in C. elegans suggest a similar role for Notch in other species that remains to be explored. Identification of Notch targets and further analysis of the cellular site of action of Notch pathway genes will help reveal the molecular mechanisms underlying quiescence and the circuitry of quiescence, respectively.

KA Kuznicki et al. (1999) International C. elegans Meeting "Unwinding the P granules: evidence from RNAi and glh mutants"

KL Bennett, JW Kirchner, PA Smith, KA Kuznicki

[International C. elegans Meeting, 1999]

The Bennett laboratory studies the four g erm l ine RNA h elicases (GLHs) , components of the C. elegans P granules. The GLHs are members of the DEAD-box family of RNA helicases and contain retroviral-like CCHC zinc fingers and uncharged glycine-rich repeats (Roussell and Bennett 1993 PNAS 90 :9300-9304, Gruidl et al . 1996 PNAS 93 :13837-13842). GLH-3 and GLH-4, more recently discovered helicases, differ from GLH-1 and GLH-2; GLH-3 lacks the uncharged glycine-rich repeats of the others, while GLH-4 is the most unique. RNAi implies that each GLH has a different role. RNAi of glh-1 or glh-2 produces sterility in a small percentage of the F 1 generation at the restrictive temperature. By contrast, glh-3 RNAi causes smaller brood sizes in the injected worm at the restrictive temperature. Using a combination of the helicase regions of glh-1 , glh-2 , and glh-3 results in a more dramatic temperature sensitive phenotype. Approximately 75% of the F 1 generation is infertile. Other combinatorial glh RNAi injections will further determine if the glhs function synergistically. A glh-3 - strain with a 1.7 kb deletion was identified in our laboratory from a TMP/UV library we generated. Preliminary analysis of the glh-3 - strain indicates that it has a mild temperature sensitive fertility defect, with brood size approximately 60-70% of that seen with wild type worms at 25-26degC. This result is consistent with that seen in the glh-3 RNAi experiments. Ongoing experiments include glh-4 RNAi and screening for additional glh mutants.

Kreutzer MA et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF THE CAENORHABDITIS ELEGANS CYCLIN GENES"

Richards JP, Bennett KL, Kreutzer MA

[International C. elegans Meeting, 1995]

The Bennett laboratory has identified genes that may be essential for nematode germline determination. Drosophila cyclin B RNA has been shown to localize to the Drosophila polar granules, germ granules that many be equivalent to the nematode P granules. Therefore, cyclins may play a role in the development of the germline. We have cloned cDNAs for the C. elegans cyclins A1, B and B3 (Kreutzer, et al. in press). Cyclins A1 and B are most closely related to either A- or B- type cyclins of other species while cyclin B3 is a distant ancestral cyclin gene. While cyclin A1 is a member of a large A-type multigene family that corresponds to a single transcript on northern blots, cyclin B3 appears to be a single gene recognizing a single transcript. In contrast, cyclin B recognizes three transcripts with one cyclin B transcript specific for the paternal germline. Our results suggest that the largest and smallest cyclin B transcripts use different polyadenylation sites. By northern analyses, the two other cyclin B as well as the cyclin A1 and cyclin B3 transcripts are most abundant in the maternal germline. In addition, two types of 3'UTR translational regulatory elements, ACEs and NREs, are found in the 3' untranslated region of each of these genes. We are currently using probes to the cyclin genes for in situ hybridization to wild type and germline defective adult worms and embryos to determine the localization of these RNAs during germline proliferation and embryogenesis.

Te-Wen Lo et al. (2007) International Worm Meeting "C. elegans FGF Receptor Signaling Can Occur via Direct Binding of the SEM-5/Grb2 Adaptor Protein."

Michael Stern, Te-Wen Lo

[International Worm Meeting, 2007]

The components of receptor tyrosine kinase(RTK)signaling complexes help to define the specificity of the effects of their activation. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2 binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. Although SEM-5 is required for the fluid homeostasis function of EGL-15, these sites are not required for that function, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multisubstrate adaptor protein FRS2 serves to link vertebrate FGFRs to GRB2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation1 but is not required for EGL-15 function2 . Thus, unlike the vertebrate FGFRs, which require the multisubstrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly. 1Matusbara et al., 2007. Genes to Cells; 12(3): 407-420. 2see D. Bennett abstract.

Chen LS et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC ANALYSIS OF C. ELEGANS NEF-1, A MEMBER OF THE IG/FNIII SUPERFAMILY OF CELL ADHESION MOLECULES"

Bennett V, Chen LS

[International C. elegans Meeting, 1997]

A cell's identity, its interactions with other cells, and its ablity to adapt to its environment are heavily dependent on the proteins associated with its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, a family of cell adhesion molecules belonging to the Ig/FnIII superfamily was shown to bind ankyrin; these proteins include neurofascin, L1, NrCam and NgCam in vertebrates and neuroglian in Drosophila (1-6). This family of ankyrin binding CAMs is believed to be involved in neurite outgrowth, axonal fasciculation and targeting, cell migration and synaptogenesis during embryonic and postnatal vertebrate development (1-4). A member of the family has been identified in C. elegans. The gene encoding the homologue has been designated nef-1. In addition to the Ig and FnIII domains, it contains a highly conserved ankyrin binding domain. We are interested in elucidating the role of nef-1 in C. elegans development and its interactions with the C. elegans ankyrin homologue, UNC-44. Interestingly, mutations in unc-44 causes defects in axon guidance (7). 1. Sonderegger and Rathgen, 1992. J.Cell Biol. 119:1387-1394. 2. Rathgen and Jessel, 1991. Sem. of Neurosci. 3:297-307. 3. Grumet, 1991. Curr. Opin. Neurobiol. 1:370-379. 4. Hortsch and Goodman, 1991. Ann. Rev. Cell Biol. 7:505-557. 5. Davis et al., 1993. J. Biol. Chem.232:121-133 6. Davis and Bennett, 1994. J. Biol. Chem.267:18955-18972. 7. Otsuka et al., 1995. J.Cell Biol. 129: 1089-1092.

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[International Worm Meeting, 2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.

Smith PA et al. (1996) Midwest Worm Meeting "C. elegans germline RNA helicases: are they all components of the P granules?"

McCrone JS, Smith PA, Bennett KL, Kuznicki KA, Kirchner JW, Strome S, Gruidl ME

[Midwest Worm Meeting, 1996]

The glh genes are a family of putative germline RNA helicases in C. elegans. glh-1 has been characterized as a germline-specific transcript coding for a predicted protein with eight conserved RNA helicase motifs, glycine-rich imperfect repeats in the N terminus, and four retroviral-like CCHC zinc fingers (Roussell and Bennett, 1993, PNAS USA 90:9300). Antibodies against two regions of GLH-1 fusion proteins recognize the germline-specific P granules of C. elegans throughout all stages of worm development. GLH-1 protein is uniformly distributed in the cytoplasm of the oocyte and segregates with the germline precursor cell from the first embryonic cell division. By Western analysis a 78kD worm protein, the predicted size for GLH-1, is seen. The closely-related glh-2 gene is also germline-specific, maps near glh-1 and codes for a less abundant, larger mRNA. The in situ hybridization patterns of glh-1 and glh-2 differ. The presence of glh-1 RNA is associated with regions of the hermaphrodite gonad where germ cell proliferation, oogenesis, and spermatogenesis occur. glh-2 mRNA is largely confined to the region where oogenesis occurs; however, glh-2 RNA is not associated with spermatogenesis. glh-2 mRNA is also detected in young C. elegans embryos. The predicted GLH-2 protein differs from GLH-1 in having six zinc fingers and different glycine repeats. Preliminary studies with GLH-2 antibodies indicate GLH-2 is present in oocytes and in P granules. The Genome Sequencing Center has uncovered an additional region on LG1 that closely matches glh-1 and glh-2. This potential GLH-3 contains two somewhat divergent CCHC fingers, but lacks the glycine repeats. Questions remain. Are all GLH proteins localized to the P granules? Do their roles differ in the germline?