Fujiwara, M. et al. (2011) International Worm Meeting "Chemotaxis behavior is regulated by germline in C. elegans."

Akamine, T., Fujiwara, M., Sato, N., Maruyama, S., Ishihara, T.

[International Worm Meeting, 2011]

Animals adequately change their behavioral patterns according to their internal states such as the extent of sexual maturation. To examine whether some signal(s) from gonad affect on worm's behavior, we analyzed a germline-ploliferation-defective mutant, glp-1, and found that this mutant shows a mild chemotaxis defect to diacetyl, an AWA-sensed volatile attractant, but not to other odorants including other AWA-sensed odorants. Ablation of the germline precursor cells (Z2, Z3) in wild-type animals with a laser microbeam confirmed the germline effect; those animals also showed the decreased chemotactic response specifically to diacetyl. To examine directly whether signal transduction in the AWA sensory neurons is affected in the germline-defective glp-1 mutant, we observed the diacetyl-evoked calcium transients by expressing a calcium indicator, Cameleon under the AWA-specific promoter. AWA neurons, in the control animals with normally developed gonads, responded to diacetyl presentation with sharp increases in calcium levels. Interestingly, the AWA neurons in the animals without germline responded to diacetyl in the similar manner, suggesting that the behavioral difference is not due to the AWA sensitivity to diacetyl per se. To reveal the molecular mechanism of the chemotactic regulation by germline, we conducted a genome-wide RNAi screening, and found that RNAi knockdown of the genes encoding electron transport chain components and ATP synthase subunits restored the chemotactic response to diacetyl in the animals without germline, suggesting the important role of mitochondria. We are currently trying the developmental-stage specific and tissue specific RNAi analyses to address how mitochondria function is involved in the control of chemotactic behavior. We hope that further analyses will reveal the molecular and cellular mechanisms how gonad signal(s) control the olfactory response to a specific attractant.

Fujiwara M et al. (2000) West Coast Worm Meeting "Regulation of C. elegans Body Size by Sensory Cues"

Phan H, McIntire SL, Fujiwara M

[West Coast Worm Meeting, 2000]

In C. elegans, body size may be regulated by the nervous system. Lewis and Hodgkin initially reported that cilium-defective mutants such as che-2 and che-3 are smaller than wild-type animals. A smaller body size is also observed in other cilium-defective mutants and in the tax-4 mutant (tax-4 encodes a cGMP-gated channel that is necessary for chemosensation). Furthermore, DBL-1/CET-1 TGF-b, which is involved in body size regulation, is expressed in neurons. These observations suggest that if an animal cannot sense an environmental cue such as food, body size may be reduced through altered neural activity. Such regulation may be useful if a smaller body is economical. Recently Apfeld and Kenyon reported that sensory cues also regulate aging of C. elegans. In order to analyze the putative neural regulation of body size, we isolated suppressor mutants of the che-2 small body size phenotype (chb). 15,000 haploid genomes were screened, yielding 28 candidates. These suppressor mutants do not suppress the dye-filling defect of che-2, but suppress the small body size of che-2. Some of these suppressors show the same body size with or without the che-2 mutation in the background. Such mutants may result from defects downstream of sensory signals. Nine strains of this category are being mapped using the snipSNP method (Wicks and Plasterk). One of these, chb-1, maps to the left arm of chromosome IV and may be allelic with odr-9/egl-4. odr-9/egl-4 maps to the same region. chb-1 has the chemotaxis defect to diacetyl that is found in odr-9/egl-4. chb-1 and odr-9/egl-4 both have a slightly larger body than wild type. We have mapped chb-1 to a reigion that is less than 100kb and are attempting to rescue with cosmids covering this region.

Fujiwara M et al. (1995) International C. elegans Meeting "SCREENING OF MORPHOLOGICAL MUTANTS IN NERVOUS SYSTEM USING GFP MARKERS."

Ishihara T, Katsura I, Fujiwara M

[International C. elegans Meeting, 1995]

We are interested in the development of the nervous system including cell lineage, cell-migration and axonal outgrowth. To visualize neurons in living worms, we expressed a GFP protein under the control of the H20 promoter, which was obtained by promoter trapping and which gave expression in almost all neurons (See the abstract by T.Ishihara and I.Katsura). Integrated H20-GFP array enabled us to examine the position and the number of neurons and the shape of neural processes. We mutagenized a H20-GFP strain with EMS and screened 2400 genomes for worms with an abnormal neural morphology by direct observation of GFP fluorescence. We concentrated our attention on post-anal ganglia and isolated three mutants. One of them lacks PLM neurons. The mutant is paralyzed, and dies at L2 or L3 stage. Since the mutation is linked to LGX, we are suspecting that the target gene may be lin-32X, the mutants of which lack PLM, too. Another mutant has an extra GFP-expressing cell near the anus. The mutant is slightly defective in egg-laying, grows slowly, and shows abnormal dye-filling; PHA and PHB do not stain with DiO. For effective mutant-screening, we have also begun a selection with strychnine, an antagonist of glycine receptors. We are planning to examine strychnine-resistant mutants with GFP expressed under the control of the promoter of a glycine receptor gene.

Fujiwara, M. et al. (2019) International Worm Meeting "Gonadal maturation changes chemotaxis to a food-associated odorant through a guanylyl cyclase GCY-28."

Wada, H., Fujiwara, M., Ishihara, T.

[International Worm Meeting, 2019]

Many animal species change their behavior depending on their stage of development. However, the mechanisms involved in translating their developmental stage into the modifications of the neuronal circuits that underlie the behavioral changes remain unknown. In Caenorhabditis elegans, the olfactory preferences are changed over development. Larvae exhibit a weak chemotactic response to the food-associated odor diacetyl, while adults exhibit a strong response. We found that the changes depend on germline cells which proliferate dramatically during the larval stages. We have shown that the germline cells affect neuronal responses to diacetyl in a circuitry comprised of a small set of neurons including an olfactory neuron, AWA, and their downstream interneurons, AIA and AIB (Fujiwara et al., 2016). Here we show that gcy-28 mutants have a defect in the germline-dependent regulation of olfactory preferences; gcy-28 mutant did not exhibit the change in chemotaxis regardless of whether germline cells proliferate normally. According to Ca2+ imaging analyses, the loss of germline cells diminished AIB calcium responses to diacetyl stimuli in wild-type animals, while, in the gcy-28 mutant, AIB responded to diacetyl normally even when the germline is lost. gcy-28 encodes a guanylyl cyclase with an extracellular ligand binding domain, and is expressed in many tissues including the nervous system. Cell-specific rescuing experiments suggested that GCY-28 acts at least partly in the AIA interneuron to mediate the developmental regulation of olfaction. GCY-28 tagged with gfp localized at gap junctions of AIA, which makes gap junctions with AWA and ASI neurons. Interestingly, blocking the gap junctions of AIA or of ASI in wild-type animals resulted in a defect in the germline-dependent chemotactic changes. We also found that silencing of ASI by expressing the leaky potassium channel unc-103(gf) caused a defect in the germline-dependent chemotactic changes. These results suggest that GCY-28 in AIA controls the germline-dependent chemotaxis by regulating ASI activity through the gap junctions. Further analyses will reveal the mechanism how the nervous system is adjusted by internal states such as sexual maturation.

Fujiwara, M. et al. (2017) International Worm Meeting "Gonadal maturation changes chemotaxis behavior and neural processing in the olfactory circuit through the function of a guanylyl cyclase, GCY-28."

Fujiwara, M., Ishihara, T.

[International Worm Meeting, 2017]

Many animal species change their behavior depending on their stage of development. However, the mechanisms involved in translating their developmental stage into the modifications of the neuronal circuits that underlie these behavioral changes remain unknown. In Caenorhabditis elegans, the olfactory preferences are changed over development, and the changes depend on germline cells which proliferate dramatically during the larval stages. We have shown that the germline cells affect the neuronal activities of a circuitry comprised of a small set of neurons including an olfactory neuron, AWA, and their downstream interneurons, AIA and AIB. Removal experiment of either one of these neurons suggested that these neurons are required for the regulation of olfactory preferences. Here we show that gcy-28 mutants have a defect in the germline-dependent regulation of olfactory preferences; gcy-28 mutant did not exhibit the change in odor preferences regardless of whether germline cells proliferate normally. gcy-28 encodes a guanylyl cyclase with an extracellular ligand binding domain, and is expressed in many tissues including the nervous system. Cell-specific rescuing experiments suggested that GCY-28 acts in the AIA interneuron to mediate the olfactory regulation. It is possible that germline proliferation may change the neural processing through modifying GCY-28 function in AIA. However, the expression levels and the subcellular localization patterns of GCY-28, which was visualized by tagged GFP, in the AIA neurons were apparently unchanged between animals lacking germline cells and animals with intact germline cells. We are now trying to elucidate how GCY-28 affects the processing in this circuitry by neuronal calcium imaging analyses. These analyses will reveal the mechanism how the nervous system is adjusted by internal states such as sexual maturation.

M Fujiwara et al. (1999) International C. elegans Meeting "A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of sensory cilia."

T Ishihara, I Katsura, M Fujiwara

[International C. elegans Meeting, 1999]

Mutations in the C. elegans gene che-2 cause defects in the cilium structure of sensory neurons and defects in behaviors that are mediated by these neurons, such as chemotaxis and osmotic avoidance (J.A.Lewis and J.A.Hodgkin 1977, T.A Starich et al 1995). We visualized the morphology of the cilia with GFP under a promoter specific to a few sensory neurons, and traced their formation during the development of the wild-type and che-2 mutant animals. The results showed that the abnormality of cilia in che-2 mutants is due to a defect in extension and not due to degeneration. We cloned che-2 gene by the cosmid rescue method. It encodes a novel protein of 760 amino acids with four WD40 repeats in the N-terminal region. WD40 repeat is known to be involved in protein-protein interaction and formation of multiprotein complexes. che-2 ( mn395 ) which has a missense mutation in the WD40 repeats, shows mild defects in various behaviors such as chemotaxis and osmotic avoidance. The other alleles which show severer behavioral defects, were revealed to have nonsense mutations in the C-terminal region, although this region has no similarity to any known protein. The genetical analysis of over-deficiency suggests that these severer alleles are candidates of null alleles. CHE-2::GFP is expressed in almost all the ciliated sensory neurons, and localized at cilia. The expression starts at the late embryonic stage when cilia are formed and continues to the adult stage. We demonstrated that che-2 acts cell-autonomously, by expressing che-2 in a subset of sensory neurons. The function and morphology of only the neurons that expressed che-2 could be restored. We also expressed che-2 cDNA under the control of a C. elegans heat shock promoter. Expression of che-2 at the larval stage or even the adult stage was sufficient for the formation of sensory cilia although it occurs at the late embryonic stage in the normal development. Furthermore, the cilia formed in the adult stage were enough for the animal to restore the ability of osmotic avoidance.

Ardiel, Evan L. et al. (2009) International Worm Meeting "A species specific chemosensory cue can modulate body size in C. elegans."

Rankin, Catharine H., Ardiel, Evan L.

[International Worm Meeting, 2009]

Previous research on C. elegans has shown that sensory perception mutants are smaller than wild-type worms (Fujiwara et al., 2002). This suggests that sensory input from the environment regulates adult body size. But what are the relevant external cues? Work in our lab suggests that interactions with conspecifics are one source. We have found that worms reared in isolation are dwarfed compared to worms reared in colonies. Furthermore, adding another worm to the plate of an isolated worm rescues colony body size. C. briggsae are also dwarfed when reared in isolation, but the relevant conspecific cue appears to be species specific, as the presence of C. briggsae cannot rescue colony body size in an isolated C. elegans. Using sensory perception mutants with cell structure (che-2, che-3, osm-3, daf-6), signal transduction (odr-1, odr-3, tax-4, osm-9), and cell specification (ceh-36, lim-4, odr-7) defects and cell-type specific rescues, we have identified a subset of chemosensory neurons with a role in the modulation of body size in response to conspecifics. Because isolated worms exposed to the stable water-soluble or volatile chemical cues of a colony are still smaller than colony worms, we propose that a species specific contact pheromone triggers growth to a larger body size. Fujiwara, M., Segupta, P., & McIntire, S.L. (2002). Regulation of body size and behavioral state of C. elegans by sensory perception and the egl-4 cGMP-dependent protein kinase. Neuron, 366 (6), 1091-1102.

Hino, T. et al. (2015) International Worm Meeting "Odor preference is changed over development in C. elegans."

Ishihara, T., Hino, T., Fujiwara, M.

[International Worm Meeting, 2015]

Many animal species change their behavioral patterns associated with their developmental stages. The behavioral changes must be important, since animals should act appropriately depending on their developmental stage. However, the molecular mechanism, which gives rise to the behavioral changes over the development, has not been revealed. Here, we explore the mechanism of the behavioral changes during the development with C. elegans. Firstly, we have shown that C. elegans larvae show different odor preference from adult animals. The larvae showed the reduced chemotactic response to diacetyl, compared to the other odorant. The increase in the chemotactic response to diacetyl during the growth may benefit the animals to locate food, since diacetyl is known to be a metabolite of bacteria. Secondly, we examined the possibility that the difference of the odor preferences of the larvae and adults was due to the difference of their moving speeds. We analyzed the chemotaxis of several mutants with different moving speeds at the adult stage, and observed no correlation between the chemotaxis index and the moving speed. This result suggests that the change of odor preference is more likely due to the modulation in sensory circuits. Finally, to reveal the molecular mechanism, we screened for mutants whose larvae show the odor preference like adults, and isolated one candidate. Since, at the adult stage, this mutant showed the same odor preference as wild-type adult, the phenotype seemed to be larval stage specific. To identify the gene responsible for the phenotype, we carried out the linkage group analysis and mapped the mutation on LGII. Whole genome sequencing revealed ~20 sequence variants predicted to affect protein-coding genes on LGII. We are now trying to identify the responsible mutation among them. We hope to uncover the molecular mechanism which gives rise to the behavioral changes associated with the development.

Hino, T. et al. (2017) International Worm Meeting "Analyses of the molecular mechanism regulating developmental change in odor preference of C. elegans."

Fujiwara, M., Ishihara, T., Hino, T.

[International Worm Meeting, 2017]

Many animal species change their behavioral patterns according to developmental stages. The behavioral changes must be important to appropriately behave at the each developmental stage. However, the molecular mechanisms which give rise to the behavioral changes depending on the development are unclear. We have shown that C. elegans larvae show a different odor preference from adults. While larval animals exhibit a weak chemotactic response to the food-related odorant, diacetyl, adult animals exhibit a strong response. To analyze the mechanism that regulates the developmental changes in the odor preferences, first, we examined which olfactory neuron contributes to the change in odor preferences. In C. elegans, two classes of olfactory neurons, AWA and AWC, act to sense attractive odorants. Both the AWA-defective and the AWC-defective mutants showed reduced chemotaxis throughout the development. These mutants, however, still exhibited chemotactic enhancement to diacetyl at the adult stage, suggesting that both olfactory neurons and/or their downstream circuits are involved in the developmental change. Second, to reveal the molecular mechanism, we screened for mutants, whose larvae show a strong attractive response to diacetyl, and isolated one mutant candidate. At the larval stage, this mutant exhibited a higher chemotactic response to diacetyl compared to wild-type larvae, while at the adult stage this mutant exhibited the similar response as wild-type adults. To identify the gene responsible for this phenotype, we mapped the mutation on LGIII. Whole genome sequencing revealed ~10 sequence variants predicted to affect protein-coding genes on LGIII. We are now trying to further narrow down the mapped region and to identify the responsible mutation among them. Furthermore, we found that cGMP-dependent protein kinase, EGL-4, is possibly involved in the developmental change of the odor preference. The egl-4 mutants at the adult stage exhibit low chemotaxis to diacetyl, compared to wild-type adults. On the other hand, egl-4 mutants at the larval stage exhibit comparable chemotaxis to diacetyl to wild-type larvae. Thus, EGL-4 may be required for chemotactic enhancement to diacetyl to occur at adult stage. By analyzing the gene identified in our screening and egl-4, we hope to uncover the molecular mechanism which gives rise to the behavioral changes during development.

Iwase, N. et al. (2017) International Worm Meeting "Mechanisms of holding memory of butanone enhancement of C. elegans."

Ishihara, T., Fujiwara, M., Iwase, N., Sawatari, E.

[International Worm Meeting, 2017]

Forgetting is important for animals to adapt themselves to changing environments. To investigate mechanisms of the forgetting, we use an olfactory learning in C. elegans. When C. elegans is exposed to butanone under a condition with enough food, the response to butanone is enhanced to show stronger chemotaxis (butanone enhancement). When they are cultivated on food without butanone after the butanone enhancement, the chemotaxis to butanone is much weaker than that before the conditioning. This phenomenon cannot be explained as a model of simple forgetting. Therefore, we hypothesized that the old memory is overwritten by the new memory (overwriting memory). To test this hypothesis, we observed a change of chemotaxis to butanone after butanone enhancement for 20 hours by using a wild type, a mutant and interneuron ablated animals. The snt-3 mutant and the AIB interneuron ablated animal, in which chemotaxis to butanone is not weakened after butanone enhancement. In the wild type, the chemotaxis index after butanone enhancement was much lower than that before the conditioning. In addition, the chemotaxis to butanone become weak like naive animals within one day. This result suggests that this phenomenon can be considered as overwriting memory and these animals forget the overwritten memory after 20 hours. In the snt-3 mutant, the enhanced chemotaxis to butanone was slowly weakened than that of wild type. This result suggests that the snt-3 gene affects the regulation of overwriting memory. In the AIB ablated snt-3, chemotaxis to butanone after butanone enhancement was slowly weakened like AIB ablated wild-type animals and snt-3, suggest that AIB neurons and snt-3 may regulate the overwriting memory in the same pathway. Naive gcy-28 mutants show weak avoidance of butanone, whereas after the conditioning, they show strong attractive response to butanone. To reveal function of gcy-28 in overwriting memory, we observed a change of the chemotaxis to butanone on food after butanone enhancement for 20 hours by using the gcy-28 mutant. In gcy-28 mutant, the chemotaxis to butanone after butanone enhancement was weakened than that of wild type and that chemotaxis is not recovered after 20 hours. This result suggests that gcy-28 affects forgetting of the overwritten memory. Our studies on after butanone enhancement suggest that, in C. elegans, some memories can be overwritten after learning when they are exposed to new environments. Our studies will reveal its mechanism.