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We have been working on functional genomics using C. elegans as a model organism. We first used cell-type specific markers and preexisting mutants to investigate how genotype-phenotype causal relationships are regulated. With the aid of transgenic methods, we analyzed various biological processes in C. elegans. We have developed efficient methods to isolate gene knockout strains. Thousands of strains isolated this way are used by many researchers and have revealed many biological mechanisms. We have also developed methods to examine the functions of genes in a comprehensive manner by integrating transgenes into chromosomes, designing conditional knockouts, and creating balancers for lethal mutations. A combination of these biological resources and techniques will be useful to understand the functions of genes in C. elegans, which has many genes that are orthologous to those of higher organisms including humans.

Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

The central neural network optimizes avoidance behavior depending on the nociceptive stimulation intensity and is essential for survival. How the property of hub neurons that enables the selection of behaviors is genetically defined is not well understood. We show that the transcription factor unc-130 , a human FOXD3/4 ortholog, is required to optimize avoidance behavior depending on stimulus strength in Caenorhabditis elegans . unc-130 is necessary for both ON responses (calcium decreases) and OFF responses (calcium increases) in AIBs, central neurons of avoidance optimization. Ablation of predicted upstream inhibitory neurons reduces the frequency of turn behavior, suggesting that optimization needs both calcium responses. At the molecular level, unc-130 upregulates the expression of at least three genes: nca-2 , a homolog of the vertebrate cation leak channel NALCN ; glr-1 , an AMPA-type glutamate receptor; and eat-4 , a hypothetical L-glutamate transmembrane transporter in the central neurons of optimization. unc-130 shows more limited regulation in optimizing behavior than an atonal homolog lin-32 , and unc-130 and lin-32 appear to act in parallel molecular pathways. Our findings suggest that unc-130 is required for the establishment of some AIB identities to optimize avoidance behavior.

The optimization of avoidance behaviors in response to stress is an instinctual life function universally present in animals. In many sexually dimorphic animals, males exhibit higher stress resistance than females, but there have been no reports of comparative studies on stress resistance in sexually dimorphic hermaphrodites capable of reproducing alone. In the present study, we aimed to utilize a reversal/turn behavioral choice to conduct a comparative analysis of optimized avoidance behavior patterns in hermaphrodite and male Caenorhabditis elegans . We found that C. elegans males showed greater resistance to physical movement under acute stress and to lifespan reduction under chronic stress than C. elegans hermaphrodites. Interestingly, males exhibited a stronger avoidance behavior pattern known as “turn” than did the hermaphrodites, even in response to mild acute stress stimuli, to which they responded as if they had been exposed to strong stimuli. Stress conditions can lead to unsuccessful mating in C. elegans , and exaggerated stress avoidance in males may have biological significance for successful mating. This sexual dimorphism in avoidance behavior optimization was attributed to neural circuits downstream of the AIB neurons, the center of turn behavior, suggesting the presence of a novel mechanism distinct from previously reported neural and molecular mechanisms of avoidance behavior optimization.

Aging is the deterioration of physiological mechanisms that is associated with getting old. There is a link between aging and mitochondrial function. However, there is an unresolved relationship between ATP levels and aging. To address this issue, we administered febuxostat (FBX), an inhibitor of human xanthine oxidase (XO)/xanthine dehydrogenase (XDH), to C. elegans. We used C. elegans as a model to evaluate the effects of FBX and to challenge the enigma of the relationship between ATP and lifespan. In this study, we showed that FBX protects mitochondria and prevents age-related muscle deterioration in C. elegans. In addition, we showed that FBX administration could increase ATP levels without overloading the mitochondria while extending the lifespan. We also showed that the combination of FBX and an antioxidant as a protection against ROS prolongs lifespan more. We have shown that the antioxidant effects and increased ATP levels may lead to antiaging effects.

With the development of massive parallel sequencing technology, it has become easier to establish new model organisms that are ideally suited to the specific biological phenomena of interest. Considering the history of research using classical model organisms, we believe that the efficient construction and sharing of gene mutation libraries will facilitate the progress of studies using these new model organisms. Using  C. elegans , we applied the TMP/UV mutagenesis method to animals lacking function in the DNA damage response genes atm-1 and xpc-1 . This method produces genetic mutations three times more efficiently than mutagenesis of wild-type animals. Furthermore, we confirmed that the use of next-generation sequencing and the elimination of false positives through machine learning could automate the process of mutation identification with an accuracy of over 95%. Eventually, we sequenced the whole genomes of 488 strains and isolated 981 novel mutations generated by the present method; these strains have been made available to anyone who wants to use them. Since the targeted DNA damage response genes are well conserved and the mutagens used in this study are also effective in a variety of species, we believe that our method is generally applicable to a wide range of animal species.

Cancer is one of the main causes of human death. Here, we focus on the B-cell lymphoma 7 protein family member B (BCL7B) gene, an accessory subunit of the SWI/SNF chromatin-remodelling complex. To characterize the function of BCL7B, heterozygous BCL7B-deficient stomach cancer cell lines were generated with the CRISPR/Cas9 genome editing system. The comprehensive gene expression patterns were compared between parental cells and each ΔBCL7B cell line by RNA-seq. The results showed marked downregulation of immune-related genes and upregulation of stemness-related genes in the ΔBCL7B cell lines. Moreover, by ChIP-seq analysis with H3K27me3 antibody, the changes of epigenetic modification sequences were compared between parental cells and each ΔBCL7B cell line. After machine learning, we detected the centroid sequence changes, which exerted an impact on antigen presentation. The regulation of BCL7B expression in cancer cells gives rise to cancer stem cell-like characteristics and the acquisition of an immune evasion phenotype.

Duchenne muscular dystrophy (DMD) is an inherited disorder with mutations in the dystrophin gene characterized by progressive muscle degeneration and weakness. Therapy such as administration of glucocorticoids, exon skipping of mutant genes and introduction of dystrophin mini-genes have been tried, but there is no radical therapy for DMD. In this study, we used C. elegans carrying mutations in the dys-1 gene as a model of DMD to examine the effects of febuxostat (FBX). We applied FBX to dys-1 mutant animals harboring a marker for muscle nuclei and mitochondria, and found that FBX ameliorates the muscle loss. We next used a severer model dys-1; unc-22 double mutant and found the dys-1 mutation causes a weakened muscle contraction. We applied FBX and other compounds to the double mutant animals and assayed the movement. We found that the administration of FBX in combination of uric acid has the best effects on the DMD model.

The combined phenotype of thrombocytopenia accompanied by intellectual disability in patients with a de novo heterozygous mutation, i.e., p.Tyr64Cys in CDC42 , signifies a clinically recognizable novel syndrome that has been eponymized as “Takenouchi-Kosaki syndrome” (OMIM #616737). In the present study, a detailed phenotypic analysis performed for a total of five patients with Takenouchi-Kosaki syndrome revealed that intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections comprise the cardinal features of this condition. A morphologic analysis of platelets derived from three affected individuals was performed using electron microscopy. The platelets of the three patients were large and spherical in shape. Furthermore, platelet α-granules were decreased, while vacuoles were increased. We further performed a functional analysis of p.Tyr64Cys in CDC42 through CRISPR/Cas9-mediated gene editing in a Caenorhabditis elegans model. This functional analysis suggested that the mutant allele has hypomorphic effects. Takenouchi-Kosaki syndrome is clinically recognizable by the combined phenotype of intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections as well as the identification of a heterozygous de novo mutation in CDC42 , i.e., p.Tyr64Cys.

Optimization of the types and timing of avoidance behaviors depending on the intensity of a noxious stimulus is essential for survival; however, processing in the central nervous system and its developmental basis are largely unknown. Here, we report that Caenorhabditis elegans preferentially selects one of three different types of avoidance behaviors depending on the strength of the noxious stimulus. We screened 210 neuronal transcription factors using a combination of optogenetics and RNA interference methods and identified 19 candidates required for avoidance behaviors. One candidate, gene lin-32 (abnormal cell LINeage 32), which encodes an atonal homolog, is required for the neural fate determination of AIB interneurons and functions by regulating the expression of electrical and chemical synapse genes, namely, inx-1 (innexin 1) and AMPA-type ionotropic glutamate receptor glr-1. When examined by Ca imaging, AIB showed an OFF calcium increase to the noxious stimulus. The OFF calcium increase was provoked only by strong stimulation, suggesting a role for optimization of the avoidance behavior. However, lin-32 mutants showed an impaired AIB OFF calcium increase, concomitant with a reduced occurrence of the dynamic avoidance behavior called the \"omega turn\". The AIB neural responses may be transferred to downstream inter/motor neurons projecting to the neck muscles via electrical synapses comprising inx-1. Finally, we found a correlation between powerful contractions of the neck muscles and omega turns. Thus, the central regulation of the magnitude and timing of activation of the AIB interneurons optimizes the probability of omega turn depending on the stimulus context.