Yuichi Iino et al. (2001) International C. elegans Meeting "A screen for suppressors of the let-60 chemotaxis defect"

Takaaki Hirotsu, Yuichi Iino, Timothy Doyle

[International C. elegans Meeting, 2001]

We have previously shown that the ras-MAPK pathway plays an important role in olfaction in C. elegans . Both loss-of-function and gain-of-function mutants of let-60 ras showed reduced chemotaxis to odorants. We also showed that the ras-MAPK pathway is involved in plasticity of chemotaxis to odorants as well: wild type worms pre-exposed to an odorant fail to show chemotaxis to that odorant, but the let-60(lf) mutant still show positive chemotaxis after such treatment. Loss-of-function mutants of mek-2 and mpk-1 suppress the chemotaxis defect of let-60(n1046gf) , indicating that the MAPK pathway acts downstream of let-60 . However, a loss-of-function mutant of lin-1 , a gene that encodes a MAPK target transcription factor in the vulval induction pathway, does not show chemotaxis defects, suggesting that other molecules act downstream of the MPK-1 MAPK in chemotaxis. To identify the components downstream of mpk-1 , we conducted a screen for suppressors of the let-60(n1046gf) chemotaxis defect. Thirteen mutants that show improved chemotaxis to isoamylalcohol were isolated after mutagenesis of let-60(gf) . During the course of the screen, we observed that the let-60(n1046) mutant also has a lethargic phenotype. When left undisturbed on bacteria-rich plates, the let-60(gf) mutant show little spontaneous locomotion compared to the wild-type. The suppressor mutants fall in several categories. Apart from suppressing the chemotaxis defect, only some of them suppressed the multivulval phenotype of let-60(gf) ; some suppressed the lethargic phenotype; some showed a hyperactive phenotype; and some showed abnormal locomotion patterns. These phenotypes apparently behaved independently, suggesting that the suppressors may each represent partially non-overlapping functions. Through analysis of these new mutants, we hope to gain further understanding of the biological processes that the ras-MAPK pathway regulates in the nervous system of C. elegans .

Yuichi Iino (2007) International Worm Meeting "Two different mechanisms contribute to salt chemotaxis in C. elegans."

Yuichi Iino

[International Worm Meeting, 2007]

"Pirouette mechanism" has been proposed as the main strategy for chemotaxis to water-soluble chemoattractants in C. elegans. Pirouette refers to a bout of sharp turns by which worms quickly change the direction of locomotion. Pirouette occurs in a stochastic manner, but its probability is modulated by the temporal change of chemoattractant concentration. When the concentration of chemoattaractant sensed by a worm declines, it shows a higher probability of pirouette, eventually biasing the worm movement towards the source of chemoattractant (1). Although this mechanism alone can generate chemotaxis, computer simulations based on the observed values show that model worms underperform real worms, suggesting possible existence of another mechanism (1). Therefore, video tracking analysis of chemotaxis was conducted anew using NaCl as a chemoattractant, which lead to identification of a navigation mechanism different from the pirouette mechanism. During runs, worms do not go straight but tend to turn slowly to either direction, apparently in a random fashion. However, the tracking analysis shows that turning is not random but biased by the spatial gradient of chemoattractant concentration. Specifically, the average turning rate is proportional to the chemoattaractant gradient in the direction perpendicular to the direction of locomotion. This mechanism is called the weather vane mechanism; by this mechanism, worms gradually change the direction of locomotion towards the gradient peak. By computer simulations, neither pirouette mechanism alone nor weather vane mechanism alone generates as efficient chemotaxis as real worms, while combination of the two generates a strong chemotaxis, suggesting contribution of both mechanisms to real chemotaxis. These results show that worms detect both temporal and spatial changes of chemical stimuli, and by utilizing both information maximize the chance to reach the source of chemicals. Mutational or physical removal of ASE sensory neurons totally eliminates both mechanisms, suggesting that the same salt-sensing neurons mediate both mechanisms. Further analysis of underlying neural circuits is under way. (1) Pierce-Shimomura, J.T., Morse, T.M. & Lockery, S.R. The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J Neurosci 19, 9557-69 (1999).

Yuichi Iino et al. (2006) East Asia C. elegans Meeting "The Unfolded Protein Response related to protease"

Hideshi Inoue, Yuichi Iino

[East Asia C. elegans Meeting, 2006]

Dipeptidyl peptidase-IV (DPPIV) cleaves N-terminal dipeptides from proteins containing proline in the penultimate position and regulates the biological activity of natural substrates. Due to its expression in numerous cells and its involvement in various mechanisms such as adhesion, apoptosis and immune response, the complete functions of DPPIV in vivo have not yet been elucidated. C. elegans has seven genes coding for DPPIV homologous, dpfs. The purpose of this study is to evaluate the role of DPPIV like protease, mainly dpf-1 and dpf-2 that are most similar to DPPIV.We found that DPFs were expressed at DTC, and dpf-1(tm779); dpf-2(tm912) mutant showed abnormal DTC migration. In contrast, overexpression of DPF-2 under the control of a lag-2 promoter showed short gonad like a gon-1 phenotype. In addition, dpf-1(tm779); dpf-2(tm912); gon-1(RNAi) suppressed the gon-1 phenotype. In a previous study, it was reported that gon-1 is expressed at DTC and body wall muscle, and abnormal DTC structure was observed in gon-1 mutant. We investigated which organelles had been influenced by gon-1(RNAi). As a result, when endoplasmic reticulum was labeled with C31E10.7::venus under the control of the lag-2 promoter, the fluorescence pattern was changed. We performed RNAi against gon-1 in the arIs37 and the transgenic animals carrying Plag-2::ssVenus. It showed inhibition of Fluorescent Protein secretion from DTC and muscle. In addition, gon-1(RNAi) induced xbp-1 mRNA splicing and increased transcription of hsp-4. These results suggest that gon-1(RNAi) induced ER stress. Furthermore, We found that down-regulation of dpf-1 and dpf-2 expression recovers ER stress.

Yuichi Iino et al. (2008) C.elegans Neuronal Development Meeting "Behavioral Strategy for Salt Chemotaxis and Underlying Neurons"

Kazushi Yoshida, Yuichi Iino, Takanobu Tagawa

[C.elegans Neuronal Development Meeting, 2008]

C. elegans shows chemotaxis to water-soluble chemoattractants such as sodium chloride. How this behavior is achieved was previously addressed by several investigators. Among them, Jon Pierce-Shimomura analyzed the behavior of animals during chemotaxis using computerized worm-tracking system and proposed a pirouette strategy (1). Worms often change the direction of locomotion by a stereotyped behavior called pirouette, a bout of reversals coupled to sharp turns. It was proposed that worms show pirouettes more often when the concentration of salt decreases, biasing their movement towards salt concentration peak. This response was directly tested and confirmed by applying salt concentration change to restricted worms, making this strategy widely accepted (2-4). We reexamined the chemotaxis behavior by using a worm-tracking system, and found another strategy called weather-vane strategy. We focused on the behavior during run, the period in which worms move forward without displaying pirouette. Quantitative analysis reveals that during run, worms tend to curve towards the side with higher concentration of salt. The average curving rate is roughly proportional to the geometrical gradient of salt concentration in the direction perpendicular to the orientation of locomotion. This strategy in principle drives the worm to orient towards the peak of salt concentration. We are further analyzing the statistical nature of the behavior. By computer simulation, neither pirouette strategy alone nor weather-vane strategy alone generates chemotaxis as efficient as real worms, but combination of the two leads to chemotaxis quantitatively similar to real worms, suggesting that worms employ the two strategies in parallel during chemotaxis. To understand the neural basis of the behavior, we laser-ablated several candidate neurons and performed worm-tracking analysis on treated animals. So far, some neurons important for both pirouette and weather-vane strategies have been identified. The neural and behavioral mechanism of chemotaxis will be discussed.

Yuichi Iino et al. (2003) International Worm Meeting "Interneuron-selective transcription factor MBR-1 is involved in olfactory plasticity"

Yu Hayashi, Takao Inoue, Takeo Kubo, Takaaki Hirotsu, Hideaki Takeuchi, Hiroyuki Arai, Hirofumi Kunitomo, Masayuki Yamamoto, Yoshihisa Mita, Yuichi Iino

[International Worm Meeting, 2003]

Processing of sensory information is mediated by specialized interneurons in various sensory systems. In insects, interneurons in the mushroom body of the brain (MB interneurons) are important for sensory integration and olfactory learning. We have previously identified the Mblk-1 gene, which encodes a novel transcription factor that is expressed preferentially in the MB interneurons of the honeybee brain.Homology searches revealed that the DNA binding motifs of Mblk-1 have significant sequence similarities with those encoded by a set of genes from nematode, fruit fly, mouse and human. However, the function of these proteins in the nervous system remains unclear. To clarify the role of Mblk-1 homologues, we performed a reverse genetic analysis using C. elegans. We identified a gene, termed mbr-1 (Mblk-1 related factor-1), which carries two DNA binding motifs with significant homologies with those of Mblk-1. mbr-1 promoter::GFP is expressed selectively in restricted sets of head interneurons identified as AIM, RIC, etc. GFP expression is first observed around the 3-fold stage and continues throughout the larval and adult stages, suggesting the possibility that mbr-1 plays a role in differentiation and/or maintenance of the function of these neurons.To investigate mbr-1 functions in sensory processing, an mbr-1 deletion mutant was isolated by the UV/TMP method and tested in chemotaxis and olfactory adaptation assays. Wild-type worms show reduced chemotaxis towards attractive odorants (benzaldehyde, isoamylalcohol and pyrazine) after pre-exposure to the same odorants for 5 min (Hirotsu and Iino, 2002 West Coast Worm Meeting). In contrast, the mbr-1 mutant worms showed a defect in the adaptation to benzaldehyde, while they showed normal plasticity with isoamylalcohol and pyrazine. The defect was rescued by a wild-type mbr-1 transgene, indicating that mbr-1 is involved in adaptation to benzaldehyde. These findings suggest that the transcription factor MBR-1 acts in specific interneuron(s) for olfactory adaptation. We are attempting cell type-specific rescues to identify the interneuron(s) where mbr-1 functions.

Kunitomo, Hirofumi et al. (2009) International Worm Meeting "A combination of salt and food conditions in the habitat affects preference for salt in C. elegans."

Kunitomo, Hirofumi, Iino, Yuichi

[International Worm Meeting, 2009]

Caenorhabditis elegans senses a wide variety of chemicals associated with food, danger, or other animals. Sodium chloride is generally considered as an attractive cue for the animal. We have previously reported, however, that the worms avoid NaCl when animals are pretreated with NaCl under starvation. This behavioral plasticity, salt chemotaxis learning, is regulated by the insulin/PI3-K signaling pathway that functions in ASER, the right member of the bilateral gustatory neurons, ASE (Tomioka et al. 2006). Here we show that cultivation of worms under trace amount of NaCl in the presence of food for several hours or longer also induces avoidance of NaCl. This type of plasticity of salt chemotaxis, tentatively called low salt conditioning, is stimulus-specific and reversible. Salt, but not osmolarity, in the growth media is required for maintaining preference of salt. To reveal the mechanisms of low salt conditioning, we assessed the behavior of the mutants that show defects in salt chemotaxis learning or in the development of specific neurons. Of the insulin signaling pathway components, age-1 and akt-1 were required for low salt conditioning, whereas ins-1 and pdk-1 were not. casy-1, an ortholog of calsyntenin essential for salt chemotaxis learning, was not involved in low salt conditioning. Interestingly, the mutants of odr-7, which is required for proper differentiation of the AWA olfactory neurons, showed severe defects in low salt conditioning but not in salt chemotaxis learning. These results indicate that salt chemotaxis is modulated by a combination of salt and food conditions in the habitat and the two plasticity paradigms, both resulting in avoidance of salt, are regulated at least in part by distinct genetic and neural mechanisms.

Kunitomo, Hirofumi et al. (2020) MicroPubl Biol

Iino, Yuichi, Kunitomo, Hirofumi

[MicroPubl Biol, 2020]

Mechanisms of chemotactic behaviors have been of great interest in C. elegans neuroscience since the early days of its research (Ward 1973). Lewis and Hodgkin (1977) systematically isolated more than ten abnormal chemotaxis (che) mutants that showed defective chemotaxis to sodium (Na+) and chloride (Cl-) ions (Lewis and Hodgkin 1977), whose responsible genes have already been molecularly characterized except for che-5(e1073). We here show that che-5(e1073) is a missense allele of gcy-22, which encodes a receptor guanylyl cyclase (rGC) specifically expressed in the ASE-right (ASER) gustatory neuron and is essential for chemosensation through the neuron.

Tomioka, Masahiro et al. (2019) International Worm Meeting "The function of axonal DAF-2c signaling in taste avoidance learning in C. elegans."

Iino, Yuichi, Tomioka, Masahiro

[International Worm Meeting, 2019]

Insulin signaling regulates brain functions and their dysfunction leads to neurological disorders. Receptors for insulin and insulin-like peptides are expressed in neurons and localize to subcellular sites, such as the plasma membrane and the axon, yet regulatory mechanisms of these signaling underlying brain functions are not fully understood. We have reported functions of splice isoforms of the C. elegans insulin receptor, DAF-2, in taste avoidance learning, in which worms learn to avoid the salt concentrations encountered under starvation. Alternative splicing of the cassette exon, E11.5, produces the exon-skipped DAF-2a and the exon-included DAF-2c isoforms. DAF-2c is transported to the axon of the ASER salt-sensing neuron under fasting and initiates learned salt avoidance through the PI3K/Akt pathway. To unveil the functions of DAF-2 signaling, we have examined genetic interactions between diacylglycerol (DAG) and DAF-2 signaling in taste avoidance learning. DAG signaling reportedly promotes attraction to high salt concentrations. After starvation conditioning, DAF-2 signaling was proposed to suppress DAG signaling at synapses of the ASER neuron, thereby suppressing high-salt attraction after starvation conditioning. Here, we show that DAF-2c signaling controls high-salt avoidance presumably through the negative regulation of the phospholipase C ? (PLC?) homolog PLC-1. Overexpression of RHO-1, whose mammalian homolog RhoA activates PLC?, caused defects in leaned high-salt avoidance similar to those seen in the DAF-2c signaling mutants. Interestingly, mutation of Ser-73 in the putative Akt phosphorylation site of RHO-1 prevented the effect of RHO-1 overexpression, implying that Akt phosphorylation may regulate DAG signaling through the control of RHO-1 in taste avoidance learning. We have been further elucidating the function of DAF-2c signaling at the neural circuit level.

Iwata, Ryo et al. (2009) International Worm Meeting "Role of the phopholipase Ce homolog PLC-1 in the regulation of salt chemotaxis learning in C. elegans."

Kunitomo, Hirofumi, Iino, Yuichi, Iwata, Ryo

[International Worm Meeting, 2009]

C. elegans shows plasticity of chemotaxis towards NaCl: when worms are starved in the presence of NaCl, their chemotaxis towards it decreases dramatically. We call this behavioral plasticity "salt chemotaxis learning". Previous works have shown the involvement of the Go-Gq signaling pathway in this process. Hyperactivation of Gqa-DAG signaling causes learning defects. As Go-Gq signaling regulates synaptic transmission at the neuromuscular junction, it is hypothesized that the learning involves changes in neurotransmitter release from the ASER salt-sensing neuron. However, it is largely unknown how past experience modifies the Go-Gq signaling and neurotransmission. Through a genetic screen for learning-defective mutants, we now identified a missense mutation pe1237 in F31B12.2, a previously uncharacterized gene predicted in Wormbase. We generated a deletion mutant pe1238, which harbors a 1.9kb deletion in the F31B12.2 gene. Intriguingly, the pe1238 mutant worms showed a behavior opposite to pe1237 and avoided the NaCl source without preconditioning. Thus, this genetic locus defines a critical regulator of salt chemotaxis learning, which determines attraction to or avoidance of NaCl. Despite the gene prediction of F31B12.2 in Wormbase, we identified a large transcript of 10kb by an RT-PCR analysis of mRNAs. This transcript spans C05A9.3, F31B12.2, frm-9 and plc-1, and encodes an in-frame fusion of the four predicted gene products. The C-terminal half of the protein encoded by the transcript is PLC-1, the homolog of the mammalian phopholipase Ce (PLCe). PLCe is an isozyme of the phospholipase C (PLC) family which contains two Ras-associating domains and a CDC25 homology domain. Small GTPases of the Ras superfamily have been suggested to activate PLCe. We are currently testing whether the large transcript functions in the regulation of salt chemotaxis learning by rescue experiments. Future work will focus on the activation mechanisms of the PLC-1 variant and how it regulates learning in combination with the Go-Gq signaling pathway.

Kunitomo, Hirofumi et al. (2010) Worm Breeder's Gazette "A modified salt chemotaxis assay to determine concentration-dependent chemotaxis"

Iino, Yuichi, Kunitomo, Hirofumi, Ohno, Hayao

[Worm Breeder's Gazette, 2010]