Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[International Worm Meeting, 2011]

To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies. 2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.

Kimura, Akatsuki (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Quantiative measurement and modeling of organelle positioning in the one-cell stage C. elegans embryo"

Kimura, Akatsuki

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The intracellular positioning of organelles and other components is important for cells to function. The one-cell stage embryo of C. elegans is a good and well-studied model to address the mechanics underlying organelle positioning. We have been focusing on the positioning of the centrosomes. By calculating the expected forces exerted by microtubules and comparing the forces with the position and velocity of the movements of centrosomes in vivo, we were able to propose feasible models to explain the positioning of the centrosomes during pronuclear centration [1], asymmetric spindle displacement [2], and spindle elongation [3]. For better understanding of the organelle positioning inside the cell, the physical properties of the cytoplasm is critical. We are starting to construct a physical model to describe dynamic behaviors of the cytoplasm. [References] 1. A. Kimura & S. Onami (2005) Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration. Dev. Cell., 8: 765-775. 2. A. Kimura & S. Onami (2007) Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans. J. Cell Biol., 179: 1347-1354. 3. Y. Hara & A. Kimura (2009) Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo. Curr. Biol., 19: 1549-1554.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Hidehito KUROYANAGI et al. (2004) East Asia Worm Meeting "SR protein kinase, SPK-1, regulates multiple aspect of post-fertilization development in C. elegans"

Hidehito KUROYANAGI, Masatoshi Hagiwara

[East Asia Worm Meeting, 2004]

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors (SR proteins), are key components of splicing machinery and are well conserved across phyla. We previously reported that SRPK in C. elegans , SPK-1, is required for germline proliferation/maturation (1,2) and embryogenesis (1). Here we report detailed analyses of SPK-1 function in embryogenesis. In order to study defects in early embryogenesis of spk-1 (RNAi) embryos we utilized time-lapse confocal imaging of wild-type or GFP-fusion protein expressing strains including histone-GFP, tubulin-GFP and PAR-2-GFP. Some spk-1 (RNAi) embryos had more than two pronuclei due to failure in releasing polar body nuclei and therefore contained more than 2n chromosomes. In most spk-1 (RNAi) embryos P0 cell divided either symmetrically or asymmetrically, in the others cytokinesis did not occur in the first cell cycle. Chromosomes were sometimes inappropriately segregated in daughter cells. In the second cleavage, a mitotic spindle in the posterior cell failed to rotate and both cells divided in transverse direction. P granules were properly localized to single posterior cell in most 2-cell or 4-cell spk-1 (RNAi) embryos, but were not restricted to single cells in later stages. Other polarity markers PAR-2-GFP and MEX-5-GFP either lost asymmetry in early one-cell stage or did not establish the asymmetry in spk-1 (RNAi) embryos. In the terminal state, the embryos consisted of many cells with little morphogenesis and frequently lacked expression of tissue-specific reporter genes. These observations suggest roles of SPK-1 in multiple aspect of early and fundamental embryogenesis. We are searching for substrate(s) of SPK-1 that regulate such processes. 1. Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K, Hagiwara M. SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development. Mech Dev. 99(1-2): 51-64, 2000. 2. Kuroyanagi H, Hagiwara M. SPK-1, a C. elegans SR protein kinase, SPK-1, is required for germline proliferation and maturation. Abstract 986, 2001 IWM.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Y Sasaki et al. (1999) International C. elegans Meeting "Differential expression of CeCRMP/DHP-1 and CeCRMP/DHP-2, novel members of CRMP/DRP/DHP/UNC-33 family in C. elegans."

T Takemoto, M Nonaka, H Kimura, Y Goshima, Y Sasaki

[International C. elegans Meeting, 1999]

Collapsin is recognized as a member of a semaphorin/collapsin gene family which is thought to be involved in axonal guidance. We isolated a collapsin response mediator protein (CRMP) from chick dorsal root ganglion, using Xenopus laevis oocyte expression system. In rodents, 4 subtypes of CRMP are expressed mainly in the nervous system during development. Independently, we cloned 4 human dihydropyrimidinase (DHP)-related proteins (DRP-1-4), human homologues of CRMP, by their homology to DHP. DHP is the second enzyme in the pyrimidine degradation pathway. However, all CRMP/DRPs have replacement at one or more essential residues for DHP activity, and CRMP/DRP purified from brain does not show any DHP activity. Therefore, a role of CRMP/DRPs in axonal guidance remains to be elucidated. Interestingly, chick CRMP/DRP shares 33% amino acid identity with the C. elegans UNC-33. unc-33 nematodes move in an uncoordinated fashion, and axon outgrowth is aberrant. In database of C. elegans Genome Project, we here identified two additional genes of CRMP/DRP/DHP/UNC-33 homologues. These two gene products shared similar amino acid identities with chick CRMP/DRP (40-46%) and rat DHP (49-55%), and thus these genes were termed CeCRMP/DHP-1 and -2 . To examine the expression of these related genes, we constructed CeCRMP/DHP::GFP (green fluorescent protein) transgenes, which were driven under control of CeCRMP/DHP native upstream genomic sequences. The CeCRMP/DHP-1::GFP was expressed in some neuronal cells and hypodermis in larval stage, but was downregulated in adult stage. On the other hand, the expression of CeCRMP/DHP-2::GFP was seen predominantly in body wall muscle cells at 180-cell stage and continued throughout life.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.

Chu, Yu-De et al. (2013) International Worm Meeting "An RNAi-based screen for the DExD/H-box RNA helicases involved in Caenorhabditis elegans microRNA function."

Chan, Shih-Peng, Chen, Shin-Kai, Chu, Yu-De, Chen, Guan-Rong, Huang, Tao

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).