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.

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]

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.

Kunitomo, Hirofumi et al. (2015) International Worm Meeting "Dissecting the roles of primary interneurons that regulate memory-dependent salt concentration chemotaxis."

Kunitomo, Hirofumi, Satoh, Yohsuke, Iino, Yuichi, Sato, Hirofumi

[International Worm Meeting, 2015]

Mapping the neuronal components of perception, memory, and motor control is critical in understanding how memory-dependent behavior is encoded by the nervous system. Salt chemotaxis of Caenorhabditis elegans is a memory-dependent navigation behavior: animals are attracted to the salt concentration at which they have been fed, whereas they avoid it if they have been starved (salt concentration chemotaxis). The animals employ a biased random walk strategy (also called the pirouette strategy) to migrate towards preferred directions. In this strategy, the frequency of reorientation depends on the temporal change of salt concentration and this bias is modulated by the differences in salt concentration between the current environment and the memorized one. Interestingly, input from a single sensory neuron, ASER, is required and sufficient for salt concentration chemotaxis under well fed conditions, and ASER is activated by salt concentration decrease irrespective of cultivation salt concentrations. To understand how sensory inputs are translated into behavior, we examined the roles of postsynaptic neurons of ASER by ablating them individually or in combination. ASER densely synapses on three pairs of interneurons, AIA, AIB and AIY. Loss of AIY or mutations in ttx-3 resulted in impaired chemotaxis to low salt but not to high salt. On the other hand, AIA-ablated animals showed a weak but significant defect in chemotaxis to high salt. Ablating both AIY and AIA disrupted chemotaxis to both directions. Quantitative analyses of locomotion revealed that these chemotaxis defects were concomitant with altered properties of the pirouette strategy: upon salt concentration decreases, AIY-abated animals showed a defect in suppression of turning after cultivation at low salt, whereas AIA-ablated animals showed a defect in activation of turning after cultivation at high salt. Ablation of AIB interneurons did not cause discernible effect on salt concentration chemotaxis, although these neurons were involved in the regulation of ASER-evoked reorientation. These results indicate that although both AIY and AIA are involved in the regulation of turning frequency upon salt concentration decreases, they differently mediate transmission of sensory information after cultivation at distinct salt concentrations.

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.

Hirofumi Kunitomo et al. (2001) International C. elegans Meeting "A new method of analyzing tissue-specific transcripts in Caenorhabditis elegans"

Yuichi Iino, Hirofumi Kunitomo

[International C. elegans Meeting, 2001]

The nematode C. elegans is a model organism that is ideal for the study of development, neuronal function, and other biological phenomena. The genome of this organism has been mostly sequenced, and existence of more than 19, 000 genes has been predicted. Genome-wide gene expression analysis provides a number of valuable clues to understanding biological processes. Transcripts that are rarely expressed or expressed in limited number of cells are often difficult to detect when mRNA is prepared from whole worms. We are developing a new method to purify the transcripts from an organ of interest or from small number of cells in the nematode. The poly(A) binding protein, a protein that specifically binds poly(A) RNA, was used to co-purify mRNA from worm cells. Nematode strains that express tagged poly(A) binding protein (Tg-PABP) under the control of tissue-specific promoters were generated. The poly(A) RNA transcribed in cells that harbor the Tg-PABP were then isolated by immunoprecipitation using an antibody against the tag. So far, we have found that the combination of crosslinking mRNA to Tg-PABP, immunoprecipitation with high concentrations of NaCl, and the addition of poly(A) oligonucleotides has increased the specificity of purification. We have started a comparative gene expression analysis of motor and sensory neurons using this technique.

Hirofumi Kunitomo et al. (2005) International Worm Meeting "Genome-wide gene expression profiling of ciliated sensory neurons in C. elegans."

Yuichi Iino, Jun Takayama, Hiroko Uesugi, Yuji KOHARA, Hirofumi Kunitomo

[International Worm Meeting, 2005]

Sensory neurons of C. elegans are highly diverged in their development, appearances, and functions. Such differences should be generated by cell type-specific gene expression. Identification of each set of genes that are expressed in a particular type of neurons is therefore important to get insights into the molecular basis of functional differences of the neurons. To characterize the genes expressed in sensory neurons, we compared mRNA prepared from ciliated sensory neurons to that from another subset of neurons using a cDNA microarray that represents 7,088 C. elegans genes. Preparation of mRNA from particular types of cells was achieved by the mRNA tagging method, in which poly(A) RNA was co-immunoprecipitated with an epitope-tagged poly(A) binding protein expressed by tissue-specific promoters (Roy et al. 2002). All the genes on the array were sorted by hybridization intensity ratios. Previously characterized sensory neuron-specific genes appeared enriched in higher rank orders indicating that our approach successfully enriched ciliated sensory neuron-expressed genes. To evaluate the results of microarray analysis further, expression patterns of the 15 genes with highest rank orders were examined using GFP reporters. Of these, 13 (87 %) were in fact expressed in ciliated sensory neurons. These results demonstrate that a combination of mRNA tagging and microarray analysis is an effective strategy for identification and characterization of genes expressed in subsets of neurons. Although the cDNA microarray has an advantage that the clones spotted were guaranteed being expressed in vivo, many sensory neuron-specific genes that are expressed in small numbers of cells are not represented. To identify and characterize sensory neuron-expressed genes thoroughly, we applied similarly prepared mRNA samples to the C. elegans Whole Genome Oligo Array distributed by The Genome Sequencing Center at Washington University (http://genome.wustl.edu/projects/celegans/microarray/), which carries most of the currently predicted C. elegans genes. At the meeting, comparison of the results from two different types of microarray will be presented. Our final goal is to characterize gene expression of each sensory neuron subtypes. So far, we have successfully enriched cell type-specific transcripts from AWC, ASEL, and ASER by applying mRNA tagging to these cells followed by quantification of transcripts using real-time PCR. These results indicate that our approach is applicable to genome-wide gene expression profiling of each type of sensory neurons.

Hirofumi Kunitomo et al. (2007) International Worm Meeting "dyf-11 encodes a novel component of intraflagellar transport machinery required for sensory cilia formation."

Yuichi Iino, Hirofumi Kunitomo

[International Worm Meeting, 2007]

Cilia develop at the dendritic endings of sensory neurons in C. elegans and play a critical role in perception of environmental stimuli. Assembly and maintenance of cilia depends on a conserved protein transport system called intraflagellar transport (IFT). The IFT system consists of anterograde (which carries ciliary components from the cell body to the ciliary tip) and retrograde (which returns turnover products from the ciliary tip to the cell body) motor complexes associated with raft-like large protein complexes called IFT particles. It is important to uncover entire components of IFT to fully understand the molecular mechanisms of ciliary formation and function. In wild-type animals, some amphid and phasmid sensory neurons absorb fluorescent dye through the sensory cilia. At least fifteen dye-filling defective (dyf) mutations have been isolated and characterized so far. Consistent with the defects in cilium structure, many genes of this class turned out to encode components of IFT. Here we describe the characterization of dyf-11, which encodes an evolutionarily conserved protein similar to the mammalian Traf3ip1 (tumor necrosis factor receptor-associated factor 3 interacting protein 1). Expression of dyf-11 is observed in most of the ciliated sensory neurons and is regulated by DAF-19, a crucial transcription factor for ciliary genes in C. elegans. dyf-11 mutants exhibit defects in dye-filling of ciliated neurons and show abnormal chemotaxis. Visualization of ciliated neurons by expression of fluorescent protein revealed that the mutants have short cilia and aberrant projections in dendrites. The cell-specific and transient action of dyf-11 in rescue experiments indicates that DYF-11 acts cell-autonomously and that its activity is required both for formation and maintenance of sensory cilia. Fluorescent protein-tagged DYF-11 localizes to cilia and moves in both directions along the axoneme. Analysis of DYF-11 movement in bbs mutants suggested that DYF-11 associates with IFT particle complex B. Most mammalian cell types develop immotile cilia called primary cilia. It is reported that the human TRAF3IP1, also called MIP-T3, is a ubiquitously expressed microtubule-binding protein. Here we show that MIP-T3 specifically localizes to cilia in MDCK cells. DYF-11 and MIP-T3 share highest similarities in their amino-terminal region, at which MIP-T3 associates with microtubules. Interestingly, lack of this region in DYF-11 did not impair its function in cilium formation.

Hirofumi Kunitomo et al. (2002) Japanese Worm Meeting "Enrichment of tissue-specific transcripts in Caenorhabditis elegans by the targeted pull-down of poly(A) tails"

Hirofumi Kunitomo, Yuichi Iino

[Japanese Worm Meeting, 2002]

Genomic array experiments provide a means for analyzing global gene expression under a variety of conditions. However, when mRNA is prepared from whole animals, it is difficult to assess changes of gene expression in a limited number of cells because of masking by the same transcripts expressed in other cells. To solve this problem, we are developing a new method for mRNA preparation using poly(A) binding protein (PABP; 'poly(A) pull-down method'). To prepare tissue-specific transcripts, nematode strains that express an epitope-tagged PABP under the control of characterized tissue-specific promoters were generated. The poly(A) RNA expressed in cells that harbor the tagged PABP were then co-purified with tagged PABP by immunoprecipitation from whole worm lysate. To minimize undesired binding of poly(A) RNA released from surrounding tissues, poly(A) RNA was crosslinked in vivo with tagged PABP before extract preparation, and purification conditions that prevent binding of tagged PABP to free poly(A) RNA were employed. Purified RNA was then evaluated by detection of tissue-specific transcripts using RT-PCR. Tagged PABP was introduced into sensory neurons or body wall muscles under the control of promoters of che-2 or myo-3 , respectively. Poly(A) RNA was prepared from transgenic animals, and the content of the mRNA for tax-2 , unc-54 , or eft-3 was evaluated by RT-PCR. Large amount of tax-2 transcripts, which are expressed in a subset of sensory neurons, were observed in poly(A) RNA of the worms which expressed tagged PABP in sensory neurons, whereas the transcripts of unc-54 were primarily detected in RNA prepared from worms that harbor PABP in muscles. Transcripts of eft-3 were amplified equally in both. These results indicated that our method of poly(A) RNA purification could enrich tissue-specific transcripts. We are now optimizing our tissue-specific mRNA preparation protocol for application to array analyses.

Hirofumi Kunitomo et al. (2006) East Asia C. elegans Meeting "dyf-11 encodes a putative microtubule-binding protein required for sensory cilia formation"

Yuichi Iino, Hirofumi Kunitomo

[East Asia C. elegans Meeting, 2006]

Most of the dendritic endings of C. elegans sensory neurons terminate in sensory cilia, microtubule-based cellular extensions, which are critical for perception of external stimuli. Assembly and maintenance of cilia is driven by intraflagellar transport (IFT), a conserved motility process that utilizes kinesin and dynein motors to move raft-like IFT particles and cargo proteins bidirectionally along microtubule axonemes. Here we describe the characterization of the dyf-11 gene. dyf-11 mutants show defects in dye-filling of amphid and phasmid sensory neurons (Dyf phenotype) and show abnormal chemotactic behavior. Visualization of sensory neurons in dyf-11 mutants by cell-specific expression of green fluorescent protein revealed that some sensory neurons in the mutants have short cilia and abnormal posterior projections. The dyf-11 gene encodes a putative microtubule-binding protein, which is conserved in ciliated/flagellated organisms from Chlamydomonas to mammals. Expression of a dyf-11 cDNA in specific neurons in dyf-11 mutants indicated that dyf-11 acts cell-autonomously for dye-uptake. Rescue experiments using a heat-shock promoter showed that the ciliary defects can be corrected at the adult stage, indicating that the activity of DYF-11 is required for formation and maintenance of sensory cilia. Expression of dyf-11 is observed in most of the ciliated neurons and is regulated by DAF-19, a crucial transcription factor for ciliary genes. Green fluorescent protein-tagged DYF-11 is localized at the cilia and moves antero- and retrogradely along the axoneme, indicating that DYF-11 is involved in IFT. These results suggest that DYF-11 acts in concert with other ciliary proteins to assemble and maintain the ciliated sensory endings in C. elegans.