Shohei Mitani et al. (2005) International Worm Meeting "Isolation and distribution of Caenorhabditis elegans deletion mutants by National Bioresource Project in Japan."

Shohei MItani, Keiko Gengyo-Ando

[International Worm Meeting, 2005]

Deletion mutants are useful to understand molecular mechanisms underlying the various biological phenomena and to analyze genetic pathways and biochemical changes in Caenorhabditis elegans. Because of the ease to examine genotypes by PCR, conventional genetic analysis should also be expedited. Those mutants are mostly powerful to examine phenotypes in detail and in combination with transgenes. To help the international research activity of the C. elegans community, we have been screening for yet isolated mutants and distributing them to laboratories world-wide. Researchers can place new target genes via WWW on the homepage of the National Biorescource Project for C. elegans (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp). We will screen for mutants in an order as requested. We would omit the targets if the mutants have been isolated elsewhere. On the home page, we will post the status of each target gene on which process of the screening. Already isolated mutants are posted on the database by the manner searchable and sorted by gene names. Alleles in which are interested by researchers could be further inspected by clicking allele names to retrieve the information about the nature of deletion sites, primers used and brief description about the phenotype if known. Researchers who wish to receive mutants are asked to send MTA to the core facility (the Mitani lab). We would like to ask researchers who have obtained even the negative data regarding what they are interested in to send information about what have been examined. This kind of information, usually being left unpublished, must be informative before researchers begin to study related genes. Because of the financial limit so far, we have been sending mutants up to 10 without any feedback. Please send reprints to the core facility, because they are helpful to continue to help the community.

Shohei Mitani et al. (2003) International Worm Meeting "Isolation and distribution of Caenorhabditis elegans deletion mutants: a custom screening service by National Bioresource Project in Japan."

Shohei MItani, Keiko Gengyo-Ando, Satoko Hanaka

[International Worm Meeting, 2003]

Deletion mutants are useful to understand molecular mechanisms underlying the various biological phenomena and to analyze genetic pathways and biochemical changes in Caenorhabditis elegans. Those mutants are mostly powerful to examine phenotypes in detail and in combination with transgenes. To help the international research activity of the C. elegans community, we started to set up a screening and distributing service system by the support of Ministry of Education, Culture, Science, Sports and Technology of Japan. We are now ready to isolate and distribute mutants by requests from research community. Researchers can add new target genes via WWW on the homepage of the National Biorescource Project for C. elegans. We will screen for mutants in an order as requested. However, to decrease duplicated effort, please avoid targets that have been in queue on homepage of other public KO projects. We would omit the targets if the mutants have been isolated elsewhere. On the home page, we will post the status of each target gene on which process of the screening, so that mutants which would be isolated soon could be visible to the researchers. In the cases, that the targets are very difficult to knockout and will take longer time, we will add the comments to the target gene list.Already isolated mutants are posted on the database by the manner searchable and sorted by gene names. Alleles in which are interested by researchers could be further inspected by the information about the nature of deletion sites, primers used and brief description about the phenotype if known. We also post laboratory by corresponding researcher names to which we have sent the mutants so that researchers can work cooperatively by interacting among laboratories by themselves. As a new trial by this project, we would like to ask researchers who have obtained even the negative data regarding what they are interested in to send information about what have been examined. This kind of information, usually being left unpublished, must be informative before researchers begin to study related genes. Such cooperative works by the community would yield more results in terms of cost-performance of the C. elegans research in near future. URL

Edgley, Mark et al. (2011) International Worm Meeting "Towards a mutation in every gene: A report from the front lines."

Barstead, Robert, Mitani, Shohei, Edgley, Mark, Moerman, Don

[International Worm Meeting, 2011]

Currently the combined efforts of the Barstead, Mitani and Moerman labs have identified over 7,000 deletions in 6,013 genes. About 1/4 of these mutants are inviable as homozygotes. Most of the mutations identified to date have been isolated using the PCR deletion detection protocol but we are also using Comparative Genome Hybridization (CGH) and massively parallel short read sequencing technologies to identify alterations. While the facilities are largely request driven, we have made a concerted effort to identify mutations in a number of large critical genes families, including transcription factors (TF's) and kinases. To date we have identified mutations in close to 900 of the 935 TF's and 381 of the 416 annotated kinases. The efforts of our three facilities when combined with efforts from the community and other more recent results from Deep Sequencing bring the total numbers of genes with either a deletion, a stop codon or a splicing defect to 8,000 genes.

HORI, Sayaka et al. (2021) International Worm Meeting "Contribution of a FOXD3/4 ortholog to optimization of avoidance behavior mediated by pre- and postsynaptic gene expression for a biphasic calcium response"

Mitani, Shohei, Hori, Sayaka

[International Worm Meeting, 2021]

The central neural network optimizes avoidance behavior depending on the nociceptive stimulation intensity is essential for survival. How the property of the hub neurons for selecting behaviors is genetically defined is not well understood. Here we show that the transcription factor unc-130, a human FOXD3/4 ortholog, is required to optimize the avoidance behavior depending on stimulus strength in Caenorhabditis elegans. unc-130 is necessary for both ON-calcium decrease and OFF-calcium increase in AIB, a central neuron of avoidance optimization. Ablation of predicted upstream inhibitory neurons reduced frequency of turn behavior, suggesting optimization needs both calcium responses. At a molecular level, unc-130 upregulates expression of at least three genes: nca-2, a homolog of the vertebrate cation leak channel NALCN, glr-1, an AMPA type glutamate receptor, and eat-4, a hypothetical L-glutamate transmembrane transporter in the central neuron of optimization. unc-130 shows more limited regulation in optimizing behavior than an atonal homolog lin-32, unc-130 and lin-32 appear to act in parallel molecular pathways. Our findings suggest that unc-130 contributes to behavioral optimization mediated by pre- and postsynaptic expression to lead biphasic neural responses.

Suehiro, Yuji et al. (2015) International Worm Meeting "Systematic revere genetics approach to identify the molecules related to choice of behaviors using Caenorhabditis elegans.."

Suehiro, Yuji, Mitani, Shohei

[International Worm Meeting, 2015]

Caenorhabditis elegans choose a behavioral goal appropriately when the worms receive conflicting sensory inputs. The neural and molecular mechanisms of the choice, however, remain unclear. To investigate the mechanisms, we performed screening of molecules related to choice of behaviors using 1498 strains carrying mutations in genes, which were predicted to be involved in neural function. As an assay to evaluate the choice, we presented two kinds of odorants diacetyl (DAc) and benzaldehyde (Bz) to the worms, both of which elicit positive chemotaxis by activating different types of sensory neurons, and counted the number of worms near the source of DAc or Bz. As a result, we found tm1811 strain carrying a deletion of metabotropic glutamate receptor MGL-1 showed excessive choice of chemotaxis toward DAc. In C. elegans, another behavioral choice assay using repellant (copper ion) and attractant (DAc) is well studied. We also tested the assay and found that tm1811 showed abnormal choice between the repellant and attractant. To investigate the behavioral pattern during our choice assay, we also recorded the motion of the worms. Then, we found that pirouettes behavior was less frequent in tm1811 rather than N2. To confirm whether MGL-1 was the responsible gene, genomic fragments including full length of MGL-1 CDS and its own promoter was injected into the tm1811. The transgenic worms showed excessive choice to Bz chemotaxis, which was opposed to the choice of tm1811. This suggests that MGL-1 is required for normal choice and the expression level of MGL-1 is crucial. MGL-1 is expressed in a subset of interneurons including AIA and AIY neurons, which regulate chemosensory behavior. To identify which neurons attribute to the choice, we generated transgenic worms expressing MGL-1 in AIA or AIY neurons of tm1811 worms. In consequence, both of transgenic strains showed excessive choice to Bz, suggesting that both of AIA and AIY were required for the normal choice. The necessity of AIA and AIY was also suggested by expression of caspase in AIA or AIY. Both of the strains expressing caspase showed excessive choice of Bz chemotaxis. Furthermore, to investigate whether the amount of MGL-1 in AIY neurons affect behavioral phenotypes, we generated three kinds of transgenic strains by injection of 1, 10 and 100 ng/microl rescue constructs. As a result, phenotypes of the strains varied depending on the concentration of injected constructs. From these data, it is hypothesized that MGL-1 inhibit the function of AIA and AIY interneurons, which suppress the pirouettes behavior, depending upon its expression level.

Yoshida, Keita et al. (2021) International Worm Meeting "Distinct pathways for exporting dsRNA in systemic RNAi"

Suehiro, Yuji, Yoshida, Keita, Yoshina, Sawako, Mitani, Shohei

[International Worm Meeting, 2021]

In C. elegans, ingestion of double stranded RNA (dsRNA) can induce systemic RNAi. Despite widely using this phenomenon as a gene knockdown method, it has not been well understood how ingested dsRNA is transported within and exported from the intestine where dsRNA is taken up from the environment. We screened for mutants that show deficiency in the feeding RNAi and identified two novel genes in which lesions affect the intercellular transport of dsRNA. One is Y39A3CL.1 encoding a novel endomembrane-associated protein that has no homologs outside of nematodes. Then, we refer to this gene as rexd-1 (RNAi EXporting Defective). The other is tbc-3, which encodes a TBC domain-containing Rab GTPase activating protein. We found that REXD-1 and TBC-3 can act in a cell non-autonomous manner, suggesting that they promote export of dsRNA during systemic RNAi. Next, we examined genetic interactions between rexd-1, tbc-3 and sid-5, which encodes an endosome-associated protein involved in transport of dsRNA across the intestine. The tbc-3 mutation enhanced the RNAi resistance phenotype of both rexd-1 and sid-5 mutations, whereas the interaction between rexd-1 and sid-5 mutations showed relatively weak enhancement of the phenotype. These results indicate that REXD-1, TBC-3 and SID-5 act in redundant pathways in systemic RNAi. Furthermore, we found that rexd-1, tbc-3 and sid-5 triple mutants have strong resistance to RNAi triggered by ingested dsRNA except for genes expressed in the intestine. In contrast, RNAi was efficiently induced in the triple mutants when dsRNA was injected into the pseudocoelom. These results suggest that in rexd-1, tbc-3 and sid-5 triple mutants, neither uptake of dsRNA nor intracellular RNAi machinery but export of dsRNA from the intestine is strongly impaired. Despite inability to export dsRNA, transport of a yolk protein from the intestine to germ cells as well as dynamics of secretion signal-fused GFP was not affected in the rexd-1, tbc-3 and sid-5 triple mutants. Therefore, it is possible that transport of dsRNA is separately regulated from general secretion pathways. Our results revealed that REXD-1 and TBC-3, together with SID-5, constitute essential pathways for export of dsRNA from the intestine in systemic RNAi.

Matsunaga, Yohei et al. (2009) International Worm Meeting "Expression mechanism of INS-18, one of the insulin-like peptides, in C. elegans."

Kawano, Tsuyoshi, Matsunaga, Yohei, Gengyo-Ando, Keiko, Mitani, Shohei

[International Worm Meeting, 2009]

The insulin/IGF-I signaling (IIS) exists in diverse species of animals. In the nematode Caenorhabiditis elegans, IIS regulates larval diapause and adult lifespan via a unique insulin receptor-like protein, DAF-2 followed by a transcription factor DAF-16. C. elegans has putative 40 insulin-like molecules, and one of them INS-18 functions as an antagonist as reported previously. Under a diapause-inducing condition, disruption of ins-18 reduces larval diapause. In addition, under a lifespan-extending condition, the disruption cancels lifespan extension. Our interest is when and whether the gene functions in induction of diapause and extension of lifespan. To address this question, we constructed reporter gene in which gfp is connected to ins-18 in a translational fusion manner, and then introduced into C. elegans, yielding a transgenic animal. We observed GFP luminescence under a diapause-inducing or a lifespan-extending condition. Moreover, we found DAF-16 recognition sequence in an upstream region of ins-18, suggesting that the gene expression is regulated by DAF-16 downstream of DAF-2. Therefore, we investigated the expression of ins-18 under daf-16(-) conditions. In this meeting, we will show the results and propose a feedback model of regulation of ins-18 expression.

Dejima, Katsufumi et al. (2019) International Worm Meeting "An endosome-resident zinc transporter is involved in negative regulation of systemic dsRNA spreading in C. elegans."

Suehiro, Yuji, Imae, Rieko, Dejima, Katsufumi, Mitani, Shohei

[International Worm Meeting, 2019]

Double-stranded RNA (dsRNA) induces post-transcriptional gene silencing in base-paring mechanisms, a phenomenon termed RNA interference (RNAi). In C. elegans, the silencing RNA is transported between cells, and RNAi occurs systemically. Previous studies implied several endomembrane proteins predicted to function in membrane trafficking in systemic RNAi. However, the molecular mechanisms that enable dsRNA export and import through membrane trafficking machineries are still poorly understood. We previously reported that RSD-3, the endosomal protein with phospholipid-binding domain (ENTH), is required for efficient import of silencing RNA, and the rsd-3 mutants displayed an incomplete defect in systemic RNAi. Here, from a genetic screen for mutations that suppress the defective RNAi phenotype of rsd-3 mutants, we identified a zinc transporter as a suppressor gene. This zinc transporter is widely expressed, acts in a cell nonautonomous manner, and is mainly localized to late endosome. The mutant itself shows the enhanced RNAi (Eri) phenotype. We tested whether this phenotype is caused by downregulation of endogenous RNAi or upregulation of exogenous RNAi, which are antagonistic. We found that certain genes targeted by the endogenous RNAi pathway were not affected in the mutants. Instead, its null mutants showed altered RAB-11 positive vesicle sizes in intestine and LMP-1 positive vesicle sizes in coelomocytes, suggesting its cell-context dependent roles in membrane trafficking. Epistasis analysis indicated that sid-1 and sid-2 but not sid-3 or sid-5 are essential for the Eri phenotype seen in the mutants. The C. elegans genome contains 28 zinc transporters. Interestingly, mutants for all other zinc transporter genes exhibited normal response to RNAi, suggesting a unique role of this transporter on RNAi. Our data revealed the zinc transporter as a novel cellular factor functioning in regulation of systemic RNAi.

Dejima, Katsufumi et al. (2021) International Worm Meeting "Analysis of endomembrane-resident zinc transporter mutants that suppress the systemic RNAi defects of rsd-3 mutant"

Suehiro, Yuji, Dejima, Katsufumi, Imae, Rieko, Mitani, Shohei

[International Worm Meeting, 2021]

Functional RNAs including double-stranded RNA (dsRNA) modulate gene expression in a sequence-dependent manner. In C. elegans, experimentally introduced dsRNA spreads over the body and leads to RNA silencing systemically. To date, several genes acting in systemic RNAi have been genetically identified. However, cellular pathways and molecules that mediate RNA transport between cells remain largely unknown. We previously reported that RSD-3/EpsinR is involved in import of silencing RNA. In this presentation, we show an analysis of suppressors for RNAi defects in the rsd-3 mutants. Through the analysis of the suppressors, we found that a zinc transporter functions as a negative regulator for dsRNA import. Several experiments support the idea that its zinc transport activity is required for negative regulation of systemic RNAi. However, while the C. elegans genome contains 28 zinc transporters, mutants for all other zinc transporter genes exhibited a normal response to RNAi, suggesting a unique role of this transporter on RNAi or genetic redundancy. Since disruption of the suppressor gene itself showed enhanced RNAi (Eri) phenotype, we also tested whether this phenotype is caused by downregulation of endogenous RNAi or upregulation of exogenous RNAi, which are antagonistic. We found that certain genes targeted by the endogenous RNAi pathway were not affected in the mutants, excluding the possibility that it acts in the core endogenous RNAi pathway. Instead, its null mutants showed altered RAB-11 positive vesicle sizes in intestine and LMP-1, RAB-7 and 2xFYVE positive vesicle sizes in coelomocytes. From these observations, we speculate that this zinc transporter would be involved in the regulation of membrane trafficking, which in turn negatively regulate exogenous RNAi in parallel or downstream of RSD-3. Our data revealed the zinc transporter as a novel cellular factor functioning in systemic RNAi.

Ding Zhen et al. (2002) Japanese Worm Meeting "Attempt to create taupathy in C. elegans"

Gengyo-Ando Keiko, Ihara Yasuo, Mitani Shohei, Ding Zhen

[Japanese Worm Meeting, 2002]

One of the hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFT) throughout the cerebral cortex, the distribution of which exactly corresponds to the area where neuronal loss occurs. The major component of NFT is a hyperphosphorylated species of tau, a microtubule-associated protein that is involved in microtubule assembly and stabilization in the cell. Recent studies1 have shown that mutations in the tau gene are linked with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). This unusual disease is characterized by extensive neuronal loss and formation of NFT, most conspicuous in the anterior half of the brain, in contrast to AD. Accordingly, the genetics tells us that tau can kill the neuron. Although some models2,3 have provided valuable insight into the mechanisms of the neurodegeneration, it is still time-consuming to use mice. Because of the high-degree conservation of the genes between C. elegans and human, it is possible that C.elegans models address questions regarding the cellular processes underlying these diseases. Above all, C.elegans offers the advantages of very short experimental cycle. I therefore sought to create tau transgenic C.elegans lines that express tau in a specific subset of neurons (mechanosensory neuron) and in all neurons to gain insight into the molecular mechanisms and pathways involved in neuronal degeneration. Reference: 1.Hutton M. et al. Nature. 1998 Jun 18;393(6686):702-5. 2.Lewis J. et al. Nat Genet. 2000 Aug;25(4):402-5. 3.Tanemura K. et al. J Neurosci. 2002 Jan 1;22(1):133-41.