Ohno, Hayao et al. (2017) International Worm Meeting "Diacylglycerol encodes differences between past and current stimulus intensity."

Ohno, Hayao, Adachi, Takeshi, Iino, Yuichi, Sakai, Naoko

[International Worm Meeting, 2017]

C. elegans can memorize external salt concentrations and is attracted to the salt concentration at which it has been fed, whereas it avoids the previous salt concentration if it has been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). An important question is how worms compare the intensity of the past and current sensory stimuli and execute appropriate chemotactic behaviors. The plasticity of salt chemotaxis is regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway and the insulin/PI3K pathway, both of which act in the ASER taste receptor neuron. Genetic manipulations that activate or inactivate the DAG/PKC pathway result in worm migration to higher or lower salt concentrations, respectively. The insulin/PI3K pathway is essential for the avoidance of the salt concentrations associated with starvation. We have monitored the abundance of DAG in the ASER presynaptic regions, where the nPKC-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in a manner dependent on the TAX-4 CNG channel subunit and the EGL-8 phospholipase C beta . These DAG responses are largely diminished after starvation. An unbiased genetic screen and mutant analyses show that the DAG pathway acts downstream of the insulin/PI3K/Akt pathway. Our results suggest that the abundance of DAG in the ASER presynaptic region can encode differences between past and current salt concentrations and the insulin/PI3K pathway regulates the change of salt concentration preferences through modulation of the DAG dynamics.

Ohno, Hayao et al. (2011) International Worm Meeting "Axonal transport of a new DAF-2 isoform governs associative learning."

Tomioka, Masahiro, Naito, Yasuki, Ohno, Hayao, Iino, Yuichi, Kato, Shinya, Kunitomo, Hirofumi

[International Worm Meeting, 2011]

It is of great survival advantage for animals to adapt to their environment by altering their behavior. Recently, we have found that C. elegans is attracted to the NaCl concentration at which food has been previously provided, whereas it learns to avoid the experienced NaCl concentration if it has been starved. The ASER salt-sensing sensory neuron has a critical role in this behavioral plasticity. In ASER, the insulin/PI3K pathway and CASY-1 act for the avoidance of the NaCl concentration experienced during starvation. Here, we show that a novel isoform of DAF-2/insulin receptor, which is produced by alternative splicing (AS) and named DAF-2c, regulates starvation-induced learning. The AS reporter assay using fluorescent proteins (Kuroyanagi et al., 2010) suggested that daf-2c is almost exclusively produced in head neurons. DAF-2c was localized to the axon of the ASER neuron, whereas DAF-2a, an isoform identified previously, was not. Moreover, the axonal localization of DAF-2c increased in response to starvation. The expression of DAF-2c rescued the learning defect of daf-2 mutants much more effectively than that of DAF-2a, although both isoforms rescued the dauer-constitutive phenotype of daf-2 mutants to a similar extent. CASY-1 is the C. elegans homolog of Calsyntenins/Alcadeins, which are type I transmembrane proteins of the cadherin superfamily highly expressed in mammalian brain. In a suppressor screen of casy-1 mutants, two missense mutations of daf-18, the homolog of PTEN phosphatase that negatively regulates the insulin/PI3K pathway, were identified. Furthermore, we found that the axonal localization of DAF-2c was abolished in casy-1 mutants. These data imply that CASY-1 regulates starvation-induced learning through the axonal transport of the DAF-2c isoform.

Ohno, Hayao et al. (2009) International Worm Meeting "Functional analysis of CASY-1, an ortholog of Calsyntenins/Alcadeins, which is essential for multiple forms of learning."

Ikeda, Daisuke D., Ohno, Hayao, Iino, Yuichi, Kunitomo, Hirofumi

[International Worm Meeting, 2009]

Caenorhabditis elegans shows various types of behavioral plasticity elicited by sensory stimuli. In salt chemotaxis learning, a learning paradigm previously reported by us, worms learn to avoid NaCl when the tastant is presented under starvation conditions. casy-1 was identified by a genetic screen for mutants that fail to show salt chemotaxis learning. CASY-1 also has essential roles in other types of learning and information processing such as olfactory adaptation, temperature learning and sensory integration. Calsyntenins/Alcadeins, the mammalian homologs of CASY-1, are highly conserved cadherin-like type I transmembrane proteins expressed in the central nervous system. Biochemical studies have shown that Calsyntenins/Alcadeins form a tripartite complex with APP (amyloid precursor protein) and X11/X11L, and are proteolytically processed in a fashion similar to APP. Whereas a genetic association of human calsyntenin-2 and memory performance has recently been reported, molecular functions of the proteins remain largely unknown. Western blotting suggested that CASY-1 is cleaved near the transmembrane domain like its mammalian orthologs, and functional domain analysis of CASY-1 revealed that the N-terminal ectodomain is essential for salt chemotaxis learning, whereas the membrane-anchored C-terminal stump is dispensable. Of the extracellular domain, the cadherin domains are dispensable, but the LG/LNS domain alone is not sufficient. Cell-specific rescue experiments demonstrated that expression of CASY-1 in the ASER salt-sensing neuron is sufficient for salt chemotaxis learning (but not for olfactory adaptation), and expression in the AWC odorant-sensing neurons is sufficient for olfactory adaptation. These results suggest that the LG/LNS domain of CASY-1 modulates chemotaxis behavior by localizing at/near the surface of the sensory neurons that sense the chemical stimuli relevant to the behavior. To identify an interacting partner(s) of CASY-1, we performed yeast two-hybrid screens using the functionally essential LG/LNS domain of CASY-1 as a bait. So far we have identified 13 genes whose products potentially interact with the domain.

Ohno, Hayao et al. (2013) International Worm Meeting "Isoform-specific axonal translocation of a novel DAF-2 isoform regulates synaptic and behavioral plasticity."

Tomioka, Masahiro, Kunitomo, Hirofumi, Iino, Yuichi, Kato, Shinya, Ohno, Hayao, Naito, Yasuki

[International Worm Meeting, 2013]

C. elegans memorizes external salt concentrations and switches the preference for them according to food availability (Kunitomo et al., submitted). The insulin/PI3K pathway and CASY-1 acting in the salt-sensing ASER sensory neuron play essential roles in the reversal of salt concentration preferences caused by starvation. CASY-1 is the C. elegans homolog of calsyntenins/alcadeins, which are cadherin-like type I transmembrane proteins highly expressed in the nervous system. In casy-1 mutants, the axonal localization of a novel isoform of the DAF-2 insulin/IGF-1 receptor, DAF-2c, was completely abolished. Further experiments suggested that CASY-1 is involved in the axonal transport of DAF-2c by acting as a molecular linker between DAF-2c and the kinesin-1 complex. The axonal localization of DAF-2c increases in response to starvation, and this DAF-2c translocation is pivotal for the regulation of synaptic transmission from ASER and the reversal of salt concentration preferences. We further found that the Ras-MAPK pathway negatively controls the CASY-1-dependent axonal transport of DAF-2c by targeting kinesin light chain. These results suggest that the CASY-1 and kinesin-1 complex regulated by the Ras-MAPK pathway confers the functional diversity to the insulin/IGF-1 receptor gene through the isoform-specific axonal transport.

Hayao Ohno et al. (2010) East Asia Worm Meeting "C. elegans has a memory for NaCl concentrations"

Yuichi Iino, Hirofumi Kunitomo, Hayao Ohno

[East Asia Worm Meeting, 2010]

Caenorhabditis elegans responds to a variety of chemicals by approaching or avoiding them. Sodium chloride is usually considered an attractive cue for the organism. We have previously reported, however, that chemotaxis to NaCl is severely reduced when animals are exposed to the salt under starvation, while starvation on a NaCl-free medium rather enhances the behavior (Saeki et al. 2001, Tomioka et al. 2006). These results indicate that the behavior is modified by a combination of NaCl and food availability.To clarify the effect of NaCl concentrations during cultivation on the behavior, a new assay for NaCl chemotaxis was devised. In this test, animals are exposed to various concentrations of NaCl with or without food followed by a behavioral test in which animals are placed on an agar plate with steep gradient of NaCl. Using this assay, we present evidence that C. elegans has a memory for NaCl concentrations: it is attracted to the NaCl concentration at which food was previously provided. Meanwhile, animals learn to avoid the NaCl concentration experienced during starvation. Mutant analyses revealed that several extracellular signaling molecules, the calsyntenin ortholog CASY-1, a secretory protein HEN-1 and an insulin homolog INS-1 are involved in the behavioral plasticity under starved conditions. ASE gustatory neurons are required for the salt concentration memory. Input of NaCl via ASER, the right member of ASE, is sufficient for acquisition of the memory, indicating that ASER generates the NaCl concentration-dependent responses.

Kunitomo, Hirofumi et al. (2011) International Worm Meeting "Experience-dependent modulation of salt preference in C. elegans."

Iino, Yuichi, Iwata, Ryo, Kunitomo, Hirofumi, Ohno, Hayao, Sato, Hirofumi

[International Worm Meeting, 2011]

Caenorhabditis elegans is attracted to a variety of chemicals including volatile odorants and water-soluble salts, and in many cases, the behavior is modified by experience. Sodium chloride is widely considered as an attractive cue for the animals. After exposure to NaCl with starvation, however, worms now avoid the salt, indicating that preference for salt is modified by the availability of NaCl and food (Saeki et al, 2001). To further clarify how salt preference is modulated by experience, we adopted a modified chemotaxis assay: animals were exposed to defined concentrations of NaCl with or without food and challenged with a chemotaxis test at a broad range of NaCl concentration. Using this assay, we found that worms are not merely attracted to salt, but they are attracted to the salt concentration at which they have been fed. In addition, worms avoid the salt concentration that they have experienced with starvation. Therefore, the behavior on salt gradient is based on salt concentration memory and depends on the food availability they experienced. The ASE neurons, which consist of functionally asymmetric cells on the left (ASEL) and right (ASER), are known as the major gustatory neurons of this organism. Analyzing the mutants that have deficits in the function of specific sensory neurons revealed that both ASE cells are required for salt concentration-dependent behavior, although contribution of ASER is larger than that of ASEL. Cell-specific recovery of ciliary function revealed that input of NaCl only from ASER is sufficient for the experience-dependent salt preference. To elucidate how salt preference is molecularly regulated, we assessed the behavior of the previously characterized mutants that show defects in salt chemotaxis plasticity. Two phospholipid signaling pathways, the diacylglycerol (DAG)/PKC pathway and the PI3K pathway are known to be involved in the control of the navigation modes (attraction or avoidance) in NaCl chemotaxis (Tomioka et al., 2006, Adachi et al., 2010). Activation and inactivation of the DAG/PKC pathway in ASER induced attraction to and avoidance of higher concentration of NaCl, respectively, irrespective of prior experience. Mutants for the insulin pathway, ins-1/insulin, age-1/PI3K and akt-1/AKT showed navigation defects only after starved conditions: they navigated to the salt concentrations they experienced even after starvation. This result is consistent with the idea that the insulin pathway is involved in switching the direction of chemotaxis toward the NaCl concentration which they have experienced.

Sakai, Naoko et al. (2015) International Worm Meeting "TOR signaling pathway is involved in regulation of salt chemotaxis learning."

Iino, Yuichi, Tomioka, Masahiro, Ohno, Hayao, Sakai, Naoko, Kunitomo, Hirofumi

[International Worm Meeting, 2015]

Previously, we have found that C. elegans changes responses to external salt concentrations depending on food availability. When worms are cultivated on a medium that contains sodium chloride (NaCl) and bacterial food, they are attracted to the NaCl concentration at which they have been previously grown. In contrast, after exposure to NaCl under starvation conditions, they learn to avoid the NaCl concentration. This behavioral change is called "salt chemotaxis learning". We also reported that insulin/PI3 kinase pathway, a well-known molecular pathway regulating dauer formation and aging, is involved in salt chemotaxis learning. However, downstream molecules of the insulin/PI3K pathway are still unknown for salt chemotaxis learning. In this study, we aimed to find new downstream molecules of insulin/PI3 kinase pathway. We employed a candidate screen and found that the TOR signaling pathway is involved in starvation-dependent behavioral changes. TOR, or Target Of Rapamycin is a nutrient-sensitive Ser/Thr kinase controlling cell growth and aging and is highly conserved from yeast to human. TOR-specific inhibitors rapamycin, Torin1 and Torin2 inhibit normal chemotaxis learning, especially after starvation conditioning. Besides, some of TOR signaling mutants also showed defects in chemotaxis learning. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2 (TOR Complex 1 and 2). Mutants of rsks-1, a downstream molecule of TORC1, prefer higher salt concentrations than wild type N2. On the other hand, mutants of one of the components of TORC2, rict-1, and those of downstream molecules of TORC2, sgk-1 and pkc-2, showed a tendency to be attracted to lower salt concentrations than N2. These results suggest that TORC1 and TORC2 act in opposite directions to control salt chemotaxis learning. .

Dupuy, Denis (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "High-throughput screening for alternative splicing regulation factors in vivo"

DUPUY, Denis

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

The aim of this our study is to systematically identify the trans-acting elements involved i alternative splicing regulation. Fluorescent reporters provide the opportunity to observe functional effects of a gene knock-down that may have a subtle phenotype that is undetectable with traditional observations. We are using a transgenic strain carrying a two-color reporter system in which two fluorescent reporters (GFP and RFP) are respectively fused to mutually exclusive alternatively spliced exons (Ohno, et al 2008) of the let-2 gene (exons 9 and 10). This strain has been subjected to a genome-wide RNAi screen for modifiers of the alternative splicing ratio. While previous RNAi screens have mostly consisted of visual observation of the worms by microscopy, we use the COPAS-Profiler to perform multidimensional quantitative analysis on the knocked-down worm populations in 96-well format. This enables us to detect not only variation in growth and fertility but also any modification of the relative expression of the two reporters i.e. modification of the alternative splicing balance. In this set up we are able to measure, for each individual RNAi experiment, the total number of worms, their sizes distribution, as well as collect longitudinal expression profiles in two fluorescent channels. Importantly, we are able to compare expression patterns within individual animals, thus providing an endogenous control for stochastic variations in overall expression level.

Ohno, H. et al. (2015) International Worm Meeting "In vivo imaging of diacylglycerol signaling in a taste receptor neuron involved in salt-concentration memory."

Iino, Y., Ohno, H.

[International Worm Meeting, 2015]

Adaptation to environmental conditions by modifying behavior often involves simple forms of learning and memory. We have previously reported that C. elegans worms can memorize ambient salt concentrations and are attracted to the previous salt concentration at which they have been fed, whereas they avoid it if they have been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). This behavioral plasticity has been suggested to be regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway acting in a taste receptor neuron, ASER. Genetic manipulations that activate or inactivate this pathway in ASER result in worm migration to higher or lower salt concentrations, respectively (Adachi et al., Genetics, 2010; Kunitomo et al., Nat. Commun., 2013). To explore the role of DAG signaling in salt concentration memory, we have monitored the abundance of DAG in ASER of living worms. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in presynaptic regions or regions adjacent to presynapses, where a novel protein kinase C (nPKC)-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. These DAG responses are largely diminished after starvation. These results raise the possibility that the activity of DAG signaling in the presynaptic region of ASER encodes the differences between past and present salt concentrations and thereby forms an integral component of the food-associated memory.

Nagashima, T. et al. (2017) International Worm Meeting "Multiple isoforms of the DAF-16/FOXO transcription factor regulate taste avoidance learning."

Tomioka, M., Nagashima, T., Iino, Y.

[International Worm Meeting, 2017]

Most Animals can memorize various environmental conditions and alter behaviors according to past experiences. Although signaling pathways underlying learning and memory have been extensively studied using various organisms such as Drosophila and mice, it has been challenging to understand the functional role of signaling pathways in defined neurons. We focus on taste avoidance learning in C. elegans, in which animals learn to avoid salt concentrations encountered under starvation conditions. We have shown that the local action of the insulin-like signaling at the axon of the ASER gustatory neuron regulates taste avoidance learning (Ohno et al., 2014). The insulin-like signaling regulates longevity, development and several other phenomena through control of DAF-16, the FOXO transcription factor homolog. On the other hand, it remains unclear how DAF-16 functions in taste avoidance learning. To clarify the role of DAF-16 in learning and memory, we have analyzed the functional role of DAF-16 in taste avoidance learning. We found that daf-16 loss-of-function mutants show defects in taste avoidance learning: they showed defects in avoidance of high or low salt concentrations after starvation conditioning with high or low salt concentrations, respectively. It was previously reported that DAF-16 has several isoforms with different functions. Behavioral phenotypes of isoform-specific mutants and isoform-specific rescue experiments suggested that at least four DAF-16 isoforms, a-, b-, d- and f-isoforms, were functional in high-salt avoidance, while only the a-isoform of DAF-16 can support low-salt avoidance. Moreover, cell-specific rescue experiments showed that expression of DAF-16 (a-isoform) in the ASER neuron rescued the defect in high-salt avoidance but not low-salt avoidance. These results imply that the functional roles of DAF-16 might vary according to the salt concentrations. We are trying to clarify the molecular mechanism downstream of DAF-16 in taste avoidance learning.