Tadao Inoue et al. (2003) International Worm Meeting "DAF-21/HSP90 functions during gonad development"

Johji Miwa, Sonoko Kawano, Tadao Inoue, Yasunori Yamaguchi

[International Worm Meeting, 2003]

HSP90 has been believed to act as a molecular chaperone and has been shown to be necessary for some developmental processes, such as signal transduction. The daf-21 (p673) mutant in C. elegans has been identified to carry a point mutation in the hsp90 homologue gene (Birnby, 2000).We have previously reported that RNA-mediated interference directed at daf-21 resulted in embryonic or larval lethality, reduced brood size, or/and sterility, which seemed to be caused by a germline defect (MidWest Worm Meeting, 2002). In addition to those, we revealed a novel aspect of DAF-21 function by daf-21 RNAi that impairment of DAF-21 function strongly affected the gonad morphology.In C. elegans, the hermaphrodite gonad consists of two U-shaped tubular arms extended anteriorly and posteriorly, each of which possesses a cell at the distal tip, called DTC. The arms are constructed in three distinct migration phases directed by the DTCs: (1) centrifugal phase, (2) ventral-to-dorsal phase, and (3) centripetal phase. More than 70% of daf-21 (RNAi) animals showed a gonadogenesis defect with a variety of DTC migration patterns in F1 and later generations. Many of these affected animals exhibited a precocious entry into the second DTC migration phase or an extra turning of the DTCs. Furthermore, others displayed no second migration phase, only the first and third phase, thus resulting in a ventral DTC reflex, which was similar to that seen in unc-5 mutants. It has been reported that unc-5 is required for the ventral-to-dorsal migration phase. Previous studies have shown that cell-autonomous unc5 expression in DTCs is necessary and sufficient for the second migration phase. Based on these phenotypic similarities with daf-21 (RNAi), an experiment to identify the timing of unc-5 expression by the testing of unc-5::gfp in daf-21 (RNAi) is underway. In addition, the daf-21 (p673) mutant strain exhibited a temperature sensitivity for the DTC migration at 25C. Taken together, our findings suggest that DAF-21 function may be required during gonad development.

Tsuyoshi Kawano et al. (2005) International Worm Meeting "Gene-disruptions and physiological functions of C. elegans insulin-like genes Ceinsulin-1 and -2"

Yasuo Kimura, Naoya Kataoka, Tsuyoshi Kawano, Shohei MItani, Keiko Gengyo-Ando, Ikuko Tanaka, Masaji Ishiguro

[International Worm Meeting, 2005]

In C. elegans, the insulin-signaling regulates the diapause, longevity and fat metabolism via the DAF-2, a homolog of insulin/IGF-I receptor. Our search for the DAF-2 ligands revealed two insulin/IGF-like peptides named Ceinsulin-1 (INS-18) and -2 (INS-17). First, in order to obtain structural information of the peptides, we performed a computer modeling using the Discover III/Insight II. The predicted tertiary structures of the peptides were quite similar to each other, and their receptor-recognition surfaces matched significantly. Therefore, these peptides should be recognized by the same receptor. Next, to elicit the physiological function of the ins-17 and -18 genes, we disrupted the genes using the TMP/UV method. The following sib-screening yielded an ins-17 deletion mutant named tm790 and an ins-18 deletion mutant named tm339. The tm790 has deletion of approximately 0.6 kb on two exons in the F56.F3 gene and the tm339 possesses deletion about 1.4 kb corresponding to two of three exons in the T28B8.2 gene. Then, we measured the life span and dauer larva formation of the mutant animals. The tm339 exhibited a slightly extended life span, while the tm790 showed a little bit of short life span. Both of the mutant animals formed no dauer larva under a normal growth-condition. However, in the presence of roughly purified diapause-inducing pheromone, the tm339 and tm790 formed more and less dauer larva, respectively, than the wild-type animal. Gengyo-Ando, K. and Mitani, S. (2000) BBRC 269: 64-69. Kawano, T., et al. (2000) BBRC 273: 431-436. Kawano, T., el al. (2003) BBB 67: 2678-2682.

Tsuyoshi Kawano et al. (2004) East Asia Worm Meeting "Gene-disruptions of C. elegans insulin-like genes Ceinsulin-1 and -2"

Naoya Kataoka, Keiko Gengyo-Ando, Shuji Honda, Tsuyoshi Kawano, Yasuo Kimura, Shohei MItani, Ikuko Tanaka

[East Asia Worm Meeting, 2004]

Gene-disruptions of C. elegans insulin-like genes Ceinsulin-1 and -2Tsuyoshi Kawano1, Naoya Kataoka1, Ikuko Tanaka1, Yasuo Kimura1, Shuji Honda2, Keiko Gengyo-Ando3 and Shohei Mitani31Department of Biological and Environmental Chemistry, Tottori University, 4-101, Minami, Koyama-cho, Tottori 680-8553, Japan2Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan3Department of Physiology, Tokyo Women , s Medical University School of Medicine, 8-1, Kawada-cho, Shinjuku-ku. Tokyo 162-8666, Japan In C. elegans, the insulin-signaling regulates the diapause, longevity and fat metabolism via the DAF-2, a homolog of insulin/IGF-I receptors. Our search for the DAF-2 ligands revealed two insulin/IGF-like peptides named Ceinsulin-1 and -2. We will also show phenotypes of the mutants lacking the genes.At first, Ceinsulin-1::gfp and Ceinsulin-2::gfp were constructed in a translational fusion manner and introduced into wild-type animals to search the cell expressing the Ceinsulin genes. The Ceinsulin-1::gfp is expressed at several neurons in the head and tail, whereas the Ceinsulin-2::gfp is expressed at several neurons only in the head. These cells do not overlap with chemosensory neurons filling DiI. Next, to Ceinsulin-1 and -2 deletion mutants designated tm339 and tm790, respectively, were generated and isolated with the TMP/UV method followed by the sib-screening. The tm339 has deletion of approximately 1.4 kb covering two of three exons in the T28B8.2 gene. The tm790 lays approximately deletion of 0.6 kb on two exons in the F56F3.6 gene. The tm339 exhibits a slightly extended life span as our previous RNAi experiment, while the tm790 shows almost the same life span as wild-type animals. Now, we are examining effects of these gene-disruptions on dauer larva formation.Gengyo-Ando, K and Mitani, S. (2000) Biochem. Biophys. Res. Commun. 269: 64-69.Kawano, T. et al. (2000) Biochem. Biophys. Res. Commun. 273: 431-436.Kawano, T. et al. (2003) Biosci. Biotechnol.Biochem. 67: 2678-2682.

Mari Ohtani et al. (2007) International Worm Meeting "Lifespan extending activity of C. elegans secreta."

Shuji Honda, Mari Ohtani, Yasuo Kimura, Yoko Honda, Tsuyoshi Kawano

[International Worm Meeting, 2007]

In a response to unfavorable growth conditions such as overpopulation and lack of adequate diet, C. elegans ceases development and enters a growth-arrested stage, a kind of larval diapause. The substance that induces the diapause is yielded by the animal and is called dauer-inducing pheromone. The animal uses the pheromone as a chemical mediator to detect overcrowding. In 2005, Jeong and co-workers isolated daumone as a dauer-inducing pheromone and elucidated its structure.1) During our trial of isolating the pheromone, we found lifespan extending activity as well as diapause-inducing activity in a crude extract of C. elegans culture..2) To remove the daumone, the crude extract was subjected to anion-exchange chromatography. The resulting extract also induced an extended lifespan, suggesting that a substance(s) distinct from daumone has lifespan extending activity. The activity disappeared in a daf-16 mutant animal, indicating daf-16 dependency. We are going to check subcellular localization of DAF-16::GFP by the extract. 1) Jeong, P. Y., et al. (2005) Nature 433:541.2) Kawano, T., et al. (2005) Biosci. Biotechnol. Biochem. 69:2479.

Andreas Ludewig et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Ascaroside Signalling and Lifespan in C. elegans"

Andreas Ludewig, Rabia Malik, Frank Schroeder, Chirag Pungaliya, Bennett Fox

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Key developmental pathways in C. elegans appear to be regulated by several sets of endogenous small molecule signals. Recent analyses in our laboratory characterized eight C. elegans-produced glycosides of the dideoxysugar ascarylose. Among these, the three ascarosides ascr#1, ascr#2, and ascr#3 had previously been shown to induce dauer formation in C. elegans; in addition, ascr#2 and ascr#3 synergize with ascr#4 as sex pheromones (Kawano et al., 2005; Butcher et al., 2007, Srinivasan et al., in press). Identification of these endogenous compounds presents a unique opportunity by providing small-molecule tools for dissecting developmental pathways. Here, we show that ascarosides ascr#2 and ascr#3 strongly increase thermo tolerance in C. elegans. Wild type worms grown on NGM medium containing nanomolar concentrations of these ascarosides survived up to six times longer at 35degC compared to untreated controls. We also found that animals exposed to ascr#2 and ascr#3 on minimal growth medium had up to three-fold increased lifespan. Notably, 50 day old adult worms grown on minimal medium containing ascr#2 and ascr#3 carried small numbers of fully developed or over-developed embryos. When re-exposed to favourableconditions, these animals gave rise to few, but viable and reproductive progeny. We currently investigate the impact of these endogenous signaling molecules on thermotolerance and life span extension in a comprehensive set of mutant strains.

Soh, Zu et al. (2019) International Worm Meeting "Computer simulation of connectome-based neural network model of Caenorhabditis elegans to generate muscle activity patterns for locomotion."

Soh, Zu, Iino, Yuichi, Sakamoto, Kazuma, Suzuki, Michiyo, Tsuji, Toshio

[International Worm Meeting, 2019]

Caenorhabditis elegans can generate coordinated movements using only 58 motor neurons. Calcium imaging studies and connectome-based mathematical models have revealed the roles of command neurons and the importance of proprioceptive feedback in generating undulatory movements. However, the current mathematical model is limited to the simulation of forward locomotion. In this study, we constructed a connectome-based model including 58 motor neurons, command neurons, and 96 muscle cells to generate muscle activity patterns for both forward and backward movements. In the previous study, we modeled the command neurons as units that output on/off signals corresponding to forward and backward movements. The motor neurons and muscle cells were modeled using the integrate-and-fire model and connected by synaptic and electric connections based on the connectome provided by WormAtlas. The strengths of the synaptic and electric connections were adjusted using backpropagation through a time algorithm so that the muscle activity patterns for generating the forward and backward locomotion could be switched by the output of the command neurons. In this paper, we performed simulation on muscle activity generation and analyzed the effects of synaptic connection strengths. The simulation results confirmed that the command neurons can switch the propagation direction of the muscle activity when the muscle activities were fed back to the motor neurons. In addition, we found that the mean activation levels of the B-class motor neurons were higher than those of A-class motor neurons when generating a forward wave and vice versa for generating a backward wave. This observation is consistent with the experimental study by Kawano et al. (2011). Furthermore, when we uniformly reduced the synaptic strengths, both the amplitudes and frequencies of the muscle activity decreased simultaneously. The model also predicts that the local synaptic strengths affect both the amplitudes and frequencies of the whole-body motions.

Tsuyoshi Kawano et al. (2003) International Worm Meeting "Comparison of gene experssion between wild-type C. elegans and a mutant lacking the Ceinsulin-1 gene"

Shohei MItani, Keiko Gengyo-Ando, Ryoko Ishii, Tsuyoshi Kawano, Yasuo Kimura

[International Worm Meeting, 2003]

In C. elegans, the insulin-signaling regulates the diapause, longevity and fat metabolism via the DAF-2, a homolog of insulin/IGF-I receptors. Our search for the DAF-2 ligands revealed three insulin/IGF-like peptides named Ceinsulin-1, -2 and -3. In the previous meeting, we showed the RNAi against Ceinsulin-1 could elongate the life span. In this meeting, we will show the difference of gene expression of the wild-type animal and a mutant lacking part of the T28B8.2 (Ceinsulin-1) gene. A Ceinsulin-1 deletion mutant tm339 was generated and isolated with the TMP/UV method followed by the sib-screening. The isolated mutant has deletion of approximately 1.4 kb covering 2 exons. After backcrossing, the life span was measured on the NGM plate containing 5-fluoro-2-deoxyuridine. In our preliminary experiment, the mutant exhibited longer life span than wild-type animals. Mean half life of wild-type animals was 20 days, whereas that of mutant animals was 26 days. The gene expressions of wild-type animal and the mutant were compared using the cDNA subtraction with a Clontech PCR-Select cDNA Subtraction Kit. We amplified each subtracted cDNA, subcloned and sequenced. We obtained fifteen independent clones derived from the cDNA of mutant animal and 16 ones from wild-type animal. We are going to ascertain whether these genes are up-regulated or down-regulated. We will also analyze the nature of these genes whether they are relevant to life span and/or metabolism. Kawano, T. et al. (2000) BBRC 273: 431-436; Gengyo-Ando, K. and Mitani, S. (2000) BBRC 269: 64-69.

Barbier, Louis et al. (2011) International Worm Meeting "The Stomatins UNC-1 and STO-6 Function in the C. elegans Motor Circuit."

Barbier, Louis, Kawano, Taizo, Po, Michelle, Zhen, Mei

[International Worm Meeting, 2011]

The knowledge of the wiring diagram and functional tests (i.e. ablation studies) of the C. elegans nervous system has led to the identification of the core components of the motor circuit, which contains five pairs of command interneurons signaling to at least six classes of motoneurons to generate patterns of locomotion. The control of C. elegans locomotion, however, is not fully understood at the molecular and cellular level. At the molecular level, mutations in many neural genes lead to specific, quantifiable movement phenotypes, but the underlying circuit defects remain poorly understood. We have used a combined molecular genetic and calcium imaging approach to dissect the function of innexin genes in regulating different layers of this motor circuit. To this end, we have shown that UNC-7 and UNC-9 innexins form gap junctions that control the output balance between the forward and backward components of the motor circuit (Kawano et al. submitted; see also Po et al., 2010 C. elegans Neuro meeting). How these gap junctions are regulated at the molecular level is unknown. In a screen for suppressors of the unc-7(e5) innexin kinker mutant, we isolated gain-of-function alleles of two stomatin genes, unc-1 and sto-6, that allow unc-7(e5) animals to move forwards. Stomatins are a conserved family of membrane-associated proteins that regulate ion channels1. Stomatins contain a stomatin domain, oligomerization domain, and occasionally other functional motifs. Like innexin unc-7 and unc-9 loss of function mutants, unc-1(lf) results in a kinker phenotype, whereas a sto-6 frameshift deletion mutant does not display an overt locomotion phenotype. unc-1(gf) and sto-6(gf) mutants, however, display unique phenotypes. We will present studies that indicate a differential role of these two classes of stomatins in regulating C. elegans motor circuit function. 1) Salzer U, Mairhofer M and Prohaska R. Dynamic Cell Biology, 2007.

Nakajima T et al. (2000) Japanese Worm Meeting "A possible candidate of the DAF-2 ligands: structure, expression, and physiological functions"

Kawano T, Kimura Y, Ito Y, Nakajima T, Ishiguro M, Takuwa K

[Japanese Worm Meeting, 2000]

The DAF-2 protein, a C. elegans homolog of an insulin/IGF-1 receptor, is involved in dauer formation, fat metabolism, and a long life span (1). In the previous meeting, we reported two distinct genes encoding insulin/IGF-1-like peptides designated Ceinsulin-1 and Ceinsulin-2 (2). In this meeting, we will make a short talk of the Ceinsulin-1 regarding its structure, expression, and physiological functions. The precursor protein of Ceinsulin-1 consists of 95 amino acid residues and seems to include a signal peptides, B and A chains, and a so-call C peptide that could be removed out through post-translational proteolysis like the preproinsulin. First, to detect the mature peptide, a polyclonal antibody against the putative B chain was prepared and used for analyzing C. elegans protein by Western blotting. The result showed that the mature Ceinsulin-1 should consist of one polypeptide like IGFs. However, the predicted tertiary structure of Ceinsulin-1 was more similar to the crystal structure of insulin than that of IGF-1, suggesting that the Ceinsulin-1 should be a hybrid molecule of insulin and IGF-1. Next, we analyzed expression pattern of the peptide at several developmental stages using RT-PCR and Western blotting. Interestingly, the transcriptional and translational patterns were not always coincident with each other, suggesting that the peptide biosynthesis could be regulated post-transcriptionally with some degrees. Details will be shown in the meeting. Finally, an RNA-i experiment was carried out to elicit physiological functions of the Ceinsulin-1. The RNAi led to reduction of the corresponding transcript and an extended life span. The inhibition of the Ceinsulin-1 biosynthesis exhibited no drastic effects on entry into and recovery from the dauer stage. Now, the effect on fat accumulation is examined. Anyhow, other insulin/IGF-1 peptides may be involved in the dauer formation and fat metabolism. References 1. Kimura, K.D., et al. (1997) Science 227, 942-946. 2. Kawano, T., et al. (1999) Peptide Science 1998: M. Kondo (Ed.) pp117-120.

Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.