Mori A et al. (2000) Japanese Worm Meeting "Studies on protein kinases involved in gametogenasis"

Higashitani A, Takahashi H, Takanami T, Mori A

[Japanese Worm Meeting, 2000]

Protein kinases are well known to play important roles, such as cell-cycle controls, apoptosis and stress responses. A lot of genes containing protein kinase domain are identified in the genome of C. elegans. However, the biological functions of their majority are still unknown. In order to isolate novel kinases involved in meiosis and gametogenasis, at first, we selected about 100 putative kinase genes in the genome. Their expression level were examined by RT-PCR reaction with total RNA from young adult and L2 larval hermaphrodite. After that, some genes whose expression levels obviously increased in young adults were investigated by the RNA interference method. As a result, we found that T08D2.7 is required for gametogenesis. In this presentation, we will discuss the results of functions of some protein kinases.

Mori, A. et al. (2019) International Worm Meeting "Developing alternative methods for high-throughput behaviour assay."

Schafer, W., Mori, A.

[International Worm Meeting, 2019]

An animal's behaviour can be considered as the output of the neural network which controls it; thus, analysing the behaviour in detail potentially reveals individual neural functions at network level. Our research goal is to understand the function of 302 neurons at the network level. Microfluidic technologies combined with image processing techniques have greatly contributed to C. elegans researches, especially behavioural analysis, over the last decade. However, applying these methods to high-throughput behavioural assays remains challenging, with a trade-off between image resolution and experimental throughput. Thus, the best solution would be to develop a system with inexpensive, small hardware device, and still allow us to capture high-resolution images/movies and with a computational platform for precisely analysing worm's behaviour and behavioural changes, together with, applicable for high-throughput researches. We have developed custom hard- and soft-wares for computational phenotyping to analyse worm's locomotion behaviour. Our hardware device with microfluidics and commercially available computer/camera modules is cost-effective and ease to manipulate and fabricate, which allows researchers to use our systems in different research objects and easily scale up the system for high throughput experiments. This device is hand-size and can fit into temperature-controlled chamber for thermal assay. Further, this system can allow worm growth in the device and provide external stimulation, indicating that this system can record worm behaviour for a long time under various types of stimulations. Our software platform not only integrates previously developed programs for behaviour features, but also employs machine-/deep-learning methods to detect specific body parts with abnormal movement. This approach could potentially reveal causative relations between neurons and movements. Analysing time-series high-resolution images allows us to quantify not only biological rhythms such as pumping and egg lay, but also features such as physiology and development. Taken together, the combination of these analyses can lead shed a light on new function of neurons. We will discuss our latest results and applications in the conference.

Mori, K. et al. (2017) International Worm Meeting "Labeling of active neural circuits by the calcium probe CaMPARI."

Toyoshima, Y., Mori, K., Iino, Y.

[International Worm Meeting, 2017]

To understand the information processing by the nervous system, it is necessary to reveal the functional connectivity of component neurons. In C. elegans the wiring diagram of all its neurons has been reported. However, the functional networks that are used for particular behaviors such as sensory perception or goal-oriented behaviors, have not yet been fully clarified. Thus, we are currently working on mapping the functional circuits to the connectome data. Here, we constructed an experimental system to extract the functional neural circuits by using a new type of genetically encoded Ca2+ indicator called CaMPARI (1) . CaMPARI fluorescence changes from green to red irreversibly only when high calcium concentration and ultraviolet (UV) light are present simultaneously. Since this probe has been used only in other model organisms such as D. melanogaster, zebrafish and mouse, we tested whether this probe works efficiently in the neurons of C. elegans. Several CaMPARI variants which show different dissociation constant (Kd) for Ca2+ have been reported. We tested some of these variants to find which one would be suitable for this new experimental system. We used ASER neuron and NaCl downstep stimulus for the verification of the CaMPARI function in C.elegans. We next made a strain that expresses CaMPARI in all neurons. We localized CaMPARI at nuclei of each neuron so that we can identify each neuron automatically and annotate them (2). By using this strain, we labeled the neurons that are active after the decrease of NaCl concentration. Our laboratory has previously shown that pairing starvation with exposure to NaCl causes salt avoidance learning in C. elegans. We are currently searching for neurons that respond differently before and after the formation of memory by using this system. (1) Fosque et al. Labeling of active neural circuits in vivo with designed calcium integrators Science (2015) (2) Toyoshima et al. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space PLOS Computational Biology (2016)</em>

Akihiro Mori et al. (2007) International Worm Meeting "Development of a new algorithm for cis-element prediction."

Akihiro Mori, Yuji KOHARA

[International Worm Meeting, 2007]

Multi-cellular organisms need adequate regulatory systems which control to activate/inactivate transcriptional process of multiple genes spatially and temporally for proper development. In many cases, this regulation occurs through the binding of proteins to a specific region of the gene. Such protein binding sites are known as cis-regulatory elements (CRE). Despite their hypothetical importance, however, the prediction of CREs by in silico methods is still a challenging problem. To address the problem, we developed a new computer algorithm for extracting cell-/stage-specific regulatory elements of C. elegans genes. The algorithm identifies CREs that are specific to target sequences, which are usually 5-upstream region. Since the size and position of such motif are not known before analysis, we consider reducing searching spaces of regulatory regions is a key factor to overcome predictive difficulties such as too many pseudo positives. Thus, we developed an algorithm named ''filtering step'' which reduces the search space (and therefore pseudo positives) dramatically, without losing the real positives. We performed multiple benchmark tests using various sets of sequences; 1) artificial sequences in which CRE are inserted, and 2) genes that contain known CREs. The results show our filtering procedure effectively works to target sequences up to 5000 bp. We also found that our algorithm can identify combinations of CRE. To identify novel CRE, we have applied the method to the gene sets that are identified to be expressed stage/cell specifically by systematic in situ hybridization analysis in this laboratory. The candidates obtained by the prediction are currently subjected to verification by in vivo experiments.

Mori I et al. (1987) International C. elegans Meeting "regulation of germ-line transposition and excision of Tc1."

Moerman DG, Mori I, Waterston RH

[International C. elegans Meeting, 1987]

Mori H et al. (1991) International C. elegans Meeting "MOLECULAR CLONING OF C. elegans CELL CYCLE GENES."

Sternberg PW, Mori H

[International C. elegans Meeting, 1991]

We are interested in studying the regulation of cell cycle progression by signal transduction events during C. elegans postembryonic development. As the first step of this project, genes that are known to play crucial roles in cell cycle control -- cdc2 serine/threonine kinase and cdc26/string homologs --were cloned by using PCR. Using a set of degenerate oligos, a ~600 bp fragment was generated off of genomic DNA template and determined to code for a part of cdc2 homolog. This fragment was then used as a probe to: l) search for cDNA and 2) determine the physical location of the gene in the genome. Approximately 140,000 plaques from a lambdaZap cDNA library (from R. Barstead) were screened: of the five candidates thus isolated, the longest cDNA was found to contain the entire open reading frame. Partial sequence of this cDNA has revealed a high degree of sequence identity between this putative C.elegans cdc2 and other cdc2 genes. The only surprise so far has been an Ile to Val substitution in the 'PSTAIR' motif that is absolutely conserved in all cdc2 genes published to date. This gene, tentatively named ncc-l (for nematode cell cycle), was physically mapped by using the YAC grid filters to right of the cluster on III, and has subsequently been shown to reside within the region covered by cosmid T05G5. A similar approach was taken to clone the C.elegans homolog of cdc25/string. Preliminary data suggest that we have isolated partial length cDNAs that can encode a gene with significant sequence similarity to other cdc25 homologs.

Mori I et al. (1993) International C. elegans Meeting "Genes and cells important for thermotaxis of C. elegans."

Ohshima YM, Honda H, Mori I

[International C. elegans Meeting, 1993]

Mori I et al. (2000) Japanese Worm Meeting "Identification of genes for temperature sensing by reverse genetics approach"

Inada H, Mori I, Komatsu H

[Japanese Worm Meeting, 2000]

Temperature is one of the most important environmental stimuli for organisms. However, the molecular mechanisms of temperature sensing are unclear in multicellular organisms, except Caenorhabditis elegans. C. elegans has a pair of thermosensory neurons (AFD) for temperature sensing, and several genes required for the response to temperature (called thermotaxis) have been identified in C. elegans. tax-2 and tax-4, which are required for normal thermotaxis, encode subunits of cyclic nucleotide gated cation channel and are expressed in many amphid sensory neurons including AFD. TAX-2/TAX-4 cation cannel shows higher affinity to cGMP than cAMP, suggesting that cGMP acts as a major second messenger of signal transduction in temperature sensing by AFD. In C. elegans, there seem to be at least 33 guanylyl cyclase (gcy) genes encoding the enzymes required to synthesize cGMP (1). Two of such genes, gcy-8 and gcy-12, are found to be expressed in AFD. The expression of gcy-8/gfp fusion is observed specifically in AFD, and the expression of gcy-12/gfp fusion is observed in many neurons and several amphid sensory neuron including AFD and AWC. We have attempted to make animals knocked out for these gcy genes by TMP/UV method. We have obtained a gcy-12 knockout strain, gcy-12(nj10), in which about 2.2 kb region containing transmembrane domain, kinese homology domain, and large part of catalytic domain was deleted. The gcy-12(nj10) animal shows almost normal thermotaxis, but exhibits abnormality in chemotaxis to isoamyl alcohol. Normal thermotaxis in gcy-12(nj10) might be resulted from gene compensation, because another gene, gcy-8, is also expressed in AFD. We are currently making gcy-8 knockout animal. (1) Yu et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 3384-3387.

Mori I et al. (2000) Japanese Worm Meeting "Genetic and behavioral analysis of neural modulation and learning in C. elegans."

Koike M, Mori I, Mohri A

[Japanese Worm Meeting, 2000]

Animals use thermal cues, besides chemical sense, for survival and reproduction in the natural environment. On a temperature gradient, C. elegans migrates toward its cultivation temperature after well-fed experience, whereas it migrates away from the cultivation temperature after starved experience (1). Here, we closely examined thermotaxis of wild type (N2) animals in order to investigate components required for the reversal of temperature preference. We found that thermotaxis to a preferred temperature (positive thermotaxis) turned into cultivation temperature avoidance (negative thermotaxis) after well-fed animals were exposed to food-deprived condition for 2.5 hours at 17-degree and only for 20 minutes at 25-degree. By contrast, negative thermotaxis turned into positive thermotaxis after food-deprived and starved animals were cultivated with food only for 10 minutes at both 17- and 25-degrees. These results suggest that C. elegans can associate cultivation temperature with feeding state information, and then modifies behavioral outputs in response to environmental changes. As already reported for other behaviors such as pharyngeal pumping and egg laying, exogenous serotonin and octopamine mimic well-fed and starved state, respectively (2). We also obtained the evidence to suggest that exogenous serotonin and octopamine mimic well-fed and starved state, respectively, in thermotaxis. First, most animals that had been placed on unseeded plates containing serotonin at 25-degree for 3 hour were found to be conditioned to show positive thermotaxis, and most animals that had been placed on seeded plates containing octopamine at 25-degree for 3 hours were conditioned to show negative thermotaxis. Second, negative thermotaxis turned into positive thermotaxis after cultivation at least for one hour at 25-degree on unseeded plate containing serotonin (10mM). However, serotonin-related monoamine mutants, cat-1(e1111), cat-2(e1112), cat-4(e1141), bas-1(ad446) and tph-1(mg280), showed somewhat abnormal, but almost normal responses in the reversal of thermotaxis behavior. Although these results seem to be inconsistent with the importance of serotonin release in well-fed state, it is possible that such serotonin-related monoamine mutants are not completely null, and a residual serotonin activity may function in these mutants. Alternatively, serotonin could be synthesized in other metabolic pathways unrelated to the pathways affected by the mutations. We believe that this behavioral modification by feeding state provides a good biological system to study learning and memory at genetic, cellular and molecular levels. (see abstract by Koike et al.). We are grateful to G. Ruvkun, Y. Shibata and S. Takagi for serotonin-related mutants. (1)Hedgecock and Russell, 1975, PNAS 72, 4061-4065. (2)Horvitz et al., 1982, Science 216, 1-12-1014.

Akihiro Mori et al. (2006) East Asia C. elegans Meeting "New computer algorithm for motif prediction"

Yuji KOHARA, Akihiro Mori

[East Asia C. elegans Meeting, 2006]

Multi-cellular organisms needs appropriate regulatory systems which control to activate/in-activate transcriptional process of multiple genes at proper stages and in proper cells for development. In many cases, this is regulated through the binding of proteins to a specific region of the gene. Such protein binding sites are known as cis-regulatory elements or motifs. However, despite their hypothetical importance, cell- and stage-specific regulatory motifs in multi-cellular organisms remain largely unrevealed. Moreover, the prediction of motifs by in silico methods and the verification of putative motifs by experimental methods are both challenging problems. To address these problems, we developed a new computer algorithm for extracting cell-/stage-specific regulatory motifs of C. elegans genes. Since the size and position of such motif are not known before analysis, we have to search short sequences (5 bases or so) in long target sequence area (more than 1000 bases), which cause too many pseudo positives and too long computation time. Thus, we developed an algorithm named &quotfiltering step&quot which reduces the search space (and therefore pseudo positives) dramatically, without losing the real positives. We performed multiple benchmark tests using various sets of genes that contain known motifs and artificial sequences in which some motifs are inserted. The results show that this filtering procedure effectively works to identify motifs in up to 2500 base region. We also found that our algorithm can identify combinations of short motifs very effectively. To identify novel motifs, we plan to apply the method to the gene sets that are known to be expressed stage/cell specifically by systematic in situ hybridization analysis in this laboratory.