Takahito Watanabe, Sumihare Noji and Taro Mito : GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs. In TALENs: Methods and Protocols (Ralf Kuhn et al. eds.), Springer, New York, 2016.
Taro Mito, Yoshiko Inoue, S Kimura, Katsuyuki Miyawaki, Nao Niwa, Yohei Sinmyo, Hideyo Ohuchi and Sumihare Noji : Formation of new organizing regions by cooperation of hedgehog, wingless, and dpp in regeneration of the insect leg; a verification of the boundary model In Morphogenesis and pattern formation in biological systems (Sekimura et al. eds.), Springer, 2003.
Takahisa Yamashita, Takahiro Ohde, Taro Nakamura, Yoshiyasu Ishimaru, Takahito Watanabe, Sayuri Tomonari, Yuki Nakamura, Sumihare Noji and Taro Mito : Involvement of the scalloped gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect Gryllus bimaculatus., Development Growth & Differentiation, Vol.65, No.6, 348-359, 2023.
(Summary)
The acquisition of wings was a key event in insect evolution. As hemimetabolous insects were the first group to acquire functional wings, establishing the mechanisms of wing formation in this group could provide useful insights into their evolution. In this study, we aimed to elucidate the expression and function of the gene scalloped (sd), which is involved in wing formation in Drosophila melanogaster, and in Gryllus bimaculatus mainly during postembryonic development. Expression analysis showed that sd is expressed in the tergal edge, legs, antennae, labrum, and cerci during embryogenesis and in the distal margin of the wing pads from at least the sixth instar in the mid to late stages. Because sd knockout caused early lethality, nymphal RNA interference experiments were performed. Malformations were observed in the wings, ovipositor, and antennae. By analyzing the effects on wing morphology, it was revealed that sd is mainly involved in the formation of the margin, possibly through the regulation of cell proliferation. In conclusion, sd might regulate the local growth of wing pads and influence wing margin morphology in Gryllus.
Shintaro Inoue, Takahito Watanabe, Taiki Hamaguchi, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji and Taro Mito : Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus., PLoS ONE, Vol.18, No.5, 2023.
(Summary)
Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.
Yuki Nakamura, Sayuri Tomonari, Kohei Kawamoto, Takahisa Yamashita, Takahito Watanabe, Yoshiyasu Ishimaru, Sumihare Noji and Taro Mito : Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus., Developmental Biology, Vol.485, 1-8, 2022.
(Summary)
Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.
Tetsuya Bando, Misa Okumura, Yuki Bando, Marou Hagiwara, Yoshimasa Hamada, Yoshiyasu Ishimaru, Taro Mito, Eri Kawaguchi, Takeshi Inoue, Kiyokazu Agata, Sumihare Noji and Hideyo Ohuchi : Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages., Development, Vol.149, No.8, 2022.
(Summary)
Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.
Takahiro Ohde, Taro Mito and Teruyuki Niimi : A hemimetabolous wing development suggests the wing origin from lateral tergum of a wingless ancestor, Nature Communications, Vol.13, No.1, 979, 2022.
(Summary)
The origin and evolution of the novel insect wing remain enigmatic after a century-long discussion. The mechanism of wing development in hemimetabolous insects, in which the first functional wings evolved, is key to understand where and how insect wings evolutionarily originate. This study explored the developmental origin and the postembryonic dramatic growth of wings in the cricket Gryllus bimaculatus. We find that the lateral tergal margin, which is homologous between apterygote and pterygote insects, comprises a growth organizer to expand the body wall to form adult wing blades in Gryllus. We also find that Wnt, Fat-Dachsous, and Hippo pathways are involved in the disproportional growth of Gryllus wings. These data provide insights into where and how insect wings originate. Wings evolved from the pre-existing lateral terga of a wingless insect ancestor, and the reactivation or redeployment of Wnt/Fat-Dachsous/Hippo-mediated feed-forward circuit might have expanded the lateral terga.
Maki Hirata, Manita Wittayarat, Zhao Namula, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Taro Mito, Sayuri Tomonari, Fuminori Tanihara and Takeshige Otoi : Generation of mutant pigs by lipofection-mediated genome editing in embryos., Scientific Reports, Vol.11, No.1, 23806, 2021.
(Summary)
The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.
Guillem Ylla, Taro Nakamura, Takehiko Itoh, Rei Kajitani, Atsushi Toyoda, Sayuri Tomonari, Tetsuya Bando, Yoshiyasu Ishimaru, Takahito Watanabe, Masao Fuketa, Yuji Matsuoka, A Austen Barnett, Sumihare Noji, Taro Mito and G Cassandra Extavour : Insights into the genomic evolution of insects from cricket genomes., Communications Biology, Vol.4, No.1, 2021.
(Summary)
Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket Gryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricket Laupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.
(Keyword)
Animals / DNA Methylation / DNA Transposable Elements / Evolution, Molecular / Female / Genes, Insect / Genome, Insect / Gryllidae / Insecta / Male / Phylogeny / Repetitive Sequences, Nucleic Acid / Sequence Analysis, DNA
Yasuaki Tomiyama, Tsugumichi Shinohara, Mirai Matsuka, Tetsuya Bando, Taro Mito and Kenji Tomioka : The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus, Zoological Letters, Vol.6, No.1, 12-26, 2020.
(Summary)
The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.
Yoshiyasu Ishimaru, Sayuri Tomonari, Takahito Watanabe, Sumihare Noji and Taro Mito : Regulatory mechanisms underlying the specification of the pupal-homologous stage in a hemimetabolous insect, Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, Vol.374, No.1783, 20190225, 2019.
(Summary)
Juvenile hormones and the genetic interaction between the transcription factors Krüppel homologue 1 (Kr-h1) and Broad (Br) regulate the transformation of insects from immature to adult forms in both types of metamorphosis (holometaboly with a pupal stage versus hemimetaboly with no pupal stage); however, knowledge about the exact instar in which this occurs is limited. Using the hemimetabolous cricket Gryllus bimaculatus (Gb), we demonstrate that a genetic interaction occurs among Gb'Kr-h1, Gb'Br and the adult-specifier transcription factor Gb'E93 from the sixth to final (eighth) nymphal instar. Gb'Kr-h1 and Gb'Br mRNAs were strongly expressed in the abdominal tissues of sixth instar nymphs, with precocious adult moults being induced by Gb'Kr-h1 or Gb'Br knockdown in the sixth instar. The depletion of Gb'Kr-h1 or Gb'Br upregulates Gb'E93 in the sixth instar. By contrast, Gb'E93 knockdown at the sixth instar prevents nymphs transitioning to adults, instead producing supernumerary nymphs. Gb'E93 also represses Gb'Kr-h1 and Gb'Br expression in the penultimate nymphal instar, demonstrating its important role in adult differentiation. Our results suggest that the regulatory mechanisms underlying the pupal transition in holometabolous insects are evolutionarily conserved in hemimetabolous G. bimaculatus, with the penultimate and final nymphal periods being equivalent to the pupal stage. This article is part of the theme issue 'The evolution of complete metamorphosis'.
Kamenura Norio, Mayumi Sugimoto, Tamehiro Norimasa, Adachi Reiko, Sayuri Tomonari, Takahito Watanabe and Taro Mito : Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy, Molecular Immunology, Vol.106, 127-134, 2019.
(Summary)
Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (r = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (r = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.
Yoshiyasu Ishimaru, Tetsuya Bando, Hideyo Ohuchi, Sumihare Noji and Taro Mito : Bone morphogenetic protein signaling in distal patterning and intercalation during leg regeneration of the cricket, Gryllus bimaculatus, Development Growth & Differentiation, Vol.60, No.6, 377-386, 2018.
(Summary)
The cricket, Gryllus bimaculatus, is a classic model of leg regeneration following amputation. We previously demonstrated that Gryllus decapentaplegic (Gb'dpp) is expressed during leg regeneration, although it remains unclear whether it is essential for this process. In this study, double-stranded RNA targeting the Smad mathers-against-dpp homolog, Gb'mad, was used to examine the role of bone morphogenetic protein (BMP) signaling in the leg regeneration process of Gryllus bimaculatus. RNA interference (RNAi)-mediated knockdown of Gb'mad led to a loss of tarsus regeneration at the most distal region of regenerating leg segments. Moreover, we confirmed that the phenotype obtained by knockdown of Dpp type I receptor, Thick veins (Gb'tkv), closely resembled that observed for Gb'mad RNAi crickets, thereby suggesting that the BMP signaling pathway is indispensable for the initial stages of tarsus formation. Interestingly, knockdown of Gb'mad and Gb'tkv resulted in significant elongation of regenerating tibia along the proximodistal axis compared with normal legs. Moreover, our findings indicate that during the regeneration of tibia, the BMP signaling pathway interacts with Dachsous/Fat (Gb'Ds/Gb'Ft) signaling and dachshund (Gb'dac) to re-establish positional information and regulate determination of leg size. Based on these observations, we discuss possible roles for Gb'mad in the distal patterning and intercalation processes during leg regeneration in Gryllus bimaculatus.
Hiroko Awata, Ryo Wakuda, Yoshiyasu Ishimaru, Yuji Matsuoka, Kanta Terao, Satomi Katata, Yukihisa Matsumoto, Yoshitaka Hamanaka, Sumihare Noji, Taro Mito and Makoto Mizunami : Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies., Scientific Reports, Vol.6, 29696, 2016.
(Summary)
Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects.
Yoshiyasu Ishimaru, Sayuri Tomonari, Yuji Matsuoka, Takahito Watanabe, Katsuyuki Miyawaki, Tetsuya Bando, Kenji Tomioka, Hideyo Ohuchi, Sumihare Noji and Taro Mito : TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis., Proceedings of the National Academy of Sciences of the United States of America, Vol.113, No.20, 5634-5639, 2016.
(Summary)
Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.
Hiroko Awata, Takahito Watanabe, Yoshitaka Hamanaka, Taro Mito, Sumihare Noji and Makoto Mizunami : Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets, Scientific Reports, Vol.5, 15885, 2015.
(Summary)
Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory.
Yoshimasa Hamada, Tetsuya Bando, Taro Nakamura, Yoshiyasu Ishimaru, Taro Mito, Sumihare Noji, Kenji Tomioka and Hideyo Ohuchi : Regenerated leg segment patterns are regulated epigenetically by histone H3K27 methylation in the cricket Gryllus bimaculatus, Development, Vol.142, No.17, 2916-2927, 2015.
(Summary)
Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression.
Yuji Matsuoka, Tetsuya Bando, Takahito Watanabe, Yoshiyasu Ishimaru, Sumihare Noji, Aleksandar Popadic and Taro Mito : Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes., Biology Open, Vol.4, No.6, 702-709, 2015.
(Summary)
In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes.
Yoshiyasu Ishimaru, Taro Nakamura, Tetsuya Bando, Yuji Matsuoka, Hideyo Ohuchi, Sumihare Noji and Taro Mito : Involvement of dachshund and Distal-less in distal pattern formation of the cricket leg during regeneration., Scientific Reports, Vol.5, 8387, 2015.
(Summary)
Cricket nymphs have the remarkable ability to regenerate a functional leg following amputation, indicating that the regenerating blastemal cells contain information for leg morphology. However, the molecular mechanisms that underlie regeneration of leg patterns remain poorly understood. Here, we analyzed phenotypes of the tibia and tarsus (three tarsomeres) obtained by knockdown with regeneration-dependent RNA interference (rdRNAi) against Gryllus dachshund (Gb'dac) and Distal-less (Gb'Dll). We found that depletion of Gb'Dll mRNA results in loss of the tarsal segments, while rdRNAi against Gb'dac shortens the tibia at the two most distal tarsomeres. These results indicate that Gb'Dll expression is indispensable for formation of the tarsus, while Gb'dac expression is necessary for elongation of the tibia and formation of the most proximal tarsomere. These findings demonstrate that mutual transcriptional regulation between the two is indispensable for formation of the tarsomeres, whereas Gb'dac is involved in determination of tibial size through interaction with Gb'ds/Gb'ft.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Takahito Watanabe, Tetsushi Sakuma, Seiichi Oyadomari, Takashi Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems., Scientific Reports, Vol.4, 5705, 2014.
(Summary)
Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.
Hiroshi Yoshida, Tetsuya Bando, Taro Mito, Hideyo Ohuchi and Sumihare Noji : An extended steepness model for leg-size determination based on Dachsous/Fat trans-dimer system., Scientific Reports, Vol.4, 4335, 2014.
(Summary)
What determines organ size has been a long-standing biological question. Lawrence et al. (2008) proposed the steepness hypothesis suggesting that the protocadherin Dachsous/Fat (Ds/Ft) system may provide some measure of dimension to the cells in relation to the gradient. In this paper we extended the model as a means of interpreting experimental results in cricket leg regeneration. We assumed that (1) Ds/Ft trans-heterodimers or trans-homodimers are redistributed during cell division, and (2) growth would cease when a differential of the dimer across each cell decreases to a certain threshold. We applied our model to simulate the results obtained by leg regeneration experiments in a cricket model. The results were qualitatively consistent with the experimental data obtained for cricket legs by RNA interference methodology. Using our extended steepness model, we provided a molecular-based explanation for leg size determination even in intercalary regeneration and for organ size determination.
Takahito Watanabe, Sumihare Noji and Taro Mito : Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs., Methods, Vol.69, No.1, 17-21, 2014.
(Summary)
Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.
Chihiro Sato Matsumoto, Hisashi Shidara, Koji Matsuda, Taro Nakamura, Taro Mito, Yukihisa Matsumoto, Kotaro Oka and Hiroto Ogawa : Targeted gene delivery in the cricket brain, using in vivo electroporation., Journal of Insect Physiology, Vol.59, No.12, 1235-1241, 2013.
(Summary)
The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.
(Keyword)
Animals / brain / Electroporation / Gene Transfer Techniques / Gryllidae / Male
V Zeng, B Ewen-Campen, HW Horch, S Roth, Taro Mito and CG Extavour : Developmental gene discovery in a hemimetabolous insect: de novo assembly and annotation of a transcriptome for the cricket Gryllus bimaculatus., PLoS ONE, Vol.8, No.5, e61479, 2013.
(Summary)
Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.
(Keyword)
Animals / Databases, Genetic / Embryonic Development / Female / Genes, Insect / Gryllidae / High-Throughput Nucleotide Sequencing / Insect Proteins / Male / Molecular Sequence Annotation / Oogenesis / Open Reading Frames / Phylogeny / Sequence Analysis, DNA / Signal Transduction / Transcriptome
Tetsuya Bando, Yoshiyasu Ishimaru, Takuro Kida, Yoshimasa Hamada, Yuji Matsuoka, Taro Nakamura, Hideyo Ohuchi, Sumihare Noji and Taro Mito : Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus, Development, Vol.140, No.5, 959-964, 2013.
(Summary)
In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb'dome), the Janus kinase Hopscotch (Gb'hop) and the transcription factor STAT (Gb'Stat) were cloned, and RNAi was performed against these genes. Gb'dome(RNAi), Gb'hop(RNAi) and Gb'Stat(RNAi) crickets showed defects in leg regeneration. Blastema expression of Gb'cyclinE was decreased in the Gb'Stat(RNAi) cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb'fat(RNAi) or Gb'warts(RNAi) was suppressed by RNAi against Gb'Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.
Inoue Junji, Y Ueda Yuuki, Bando Tetsuya, Taro Mito, Sumihare Noji and Hideyo Ohuchi : The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation., Development Growth & Differentiation, Vol.55, No.7, 668-675, 2013.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, W Hadley Horch, Naoya Hamaguchi, Taro Nakamura, Tetsuya Bando, Hideyo Ohuchi, Takashi Yamamoto, Sumihare Noji and Taro Mito : Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases., Nature Communications, Vol.3, 1017-1025, 2012.
(Summary)
Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.
Katsuyuki Miyawaki, Sachi Fukuoka, Yasuko Kadomura, Hirokazu Hamaoka, Taro Mito, Hideyo Ohuchi, Wilfried Schwab and Sumihare Noji : Establishment of a novel system to elucidate the mechanisms underlying light-induced ripening of strawberry fruit with an Agrobacterium-mediated RNAi technique, Plant Biotechnology, Vol.29, 271-278, 2012.
(Summary)
Traditional methods used to study strawberry ripening-related gene function are time-consuming, and require at least 15 months from initiating the transformation experiment until the first ripe fruits are available for analysis. To accelerate data acquisition during gene function studies, we explored a transient assay method that employs an Agrobacterium-mediated RNAi (AmRNAi) technique in post-harvest strawberry fruit, Fragaria×ananassa (Fa) cv. Sachinoka, a Japanese cultivar. Our results showed that artificial white light induced strong expression of Fa chalcone synthase (Fa CHS), Fa chalcone isomerase (Fa CHI), and Fa flavonoid 3 -hydroxylase orthologues (Fa F3 H) in post-harvest fruit. Fa CHS and Fa F3 H function was subsequently examined by performing AmRNAi with post-harvest fruit. Although reduction of light-induced Fa F3 H expression by AmRNAi resulted in no significant change in anthocyanin content, reduction of Fa CHS significantly decreased anthocyanin levels, and up-regulated Fa F3 H levels. Our results are consistent with previous data indicating that while CHS is required for anthocyanin accumulation during late stage strawberry fruit maturation, Fa F3 H is not required. The novel system described here enabled gene function data to be available within 10 days of initiating the incubation period following infiltration. Therefore, we conclude our system is a valuable tool to elucidate the molecular mechanisms underlying light-induced ripening of strawberry fruit.
Akira Takagi, Kazuki Kurita, Taiki Terasawa, Taro Nakamura, Tetsuya Bando, Yoshiyuki Moriyama, Taro Mito, Sumihare Noji and Hideyo Ohuchi : Functional analysis of the role of eyes absent and sine oculis in the developing eye of the cricket Gryllus bimaculatus., Development Growth & Differentiation, Vol.54, No.2, 227-240, 2012.
(Summary)
In the cricket Gryllus bimaculatus, a hemimetabolous insect, the compound eyes begin to form in the embryo and increase 5-6 fold in size during the postembryonic development of the nymphal stage. Retinal stem cells in the anteroventral proliferation zone (AVPZ) of the nymphal eye proliferate to increase retinal progenitors, which then differentiate to form new ommatidia in the anterior region of the eye. However, mechanisms underlying this type of eye formation have not been well elucidated yet. Here, we found that the homologues of the retinal determination transcription factor genes of eyes absent (eya) and sine oculis (so) are expressed during the cricket embryonic eye formation. eya is also expressed intensely in the AVPZ of the nymphal eye. To explore their functions, we performed knockdown by RNA interference (RNAi). Knockdown of Gb'eya resulted in loss of the embryonic eye. In the nymphal eye, RNAi against Gb'eya or Gb'so impaired retinal morphology by apparently transforming cornea structures into head cuticle. These results imply that Gb'eya and Gb'so are essential for the differentiation of the retinal progenitor cells and maintaining retinal structures during eye development.
Taro Mito, Yohei Shinmyo, Kazuki Kurita, Taro Nakamura, Hideyo Ohuchi and Sumihare Noji : Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus., Development, Vol.138, No.17, 3823-3833, 2011.
(Summary)
Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.
(Keyword)
Animals / Body Patterning / Cell Proliferation / Embryo, Nonmammalian / Embryonic Development / Extremities / Gryllidae / Immunohistochemistry / In Situ Hybridization / Intracellular Signaling Peptides and Proteins / Membrane Proteins / RNA Interference / Receptors, Notch / Signal Transduction
Noha Dabour, Tetsuya Bando, Taro Nakamura, Katsuyuki Miyawaki, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor., Development Growth & Differentiation, Vol.53, No.7, 857-869, 2011.
(Summary)
A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.
Tetsuya Bando, Taro Mito, Taro Nakamura, Hideyo Ohuchi and Sumihare Noji : Regulation of leg size and shape:involvement of the Dachsous/Fat signaling pathway, Developmental Dynamics, Vol.240, No.5, 1028-1041, 2011.
(Summary)
How limb size and shape is regulated is a long-standing question in developmental and regeneration biology. Recently, the protocadherin Dachsous-Fat (Ds-Ft) signaling pathway has been found to be essential for wing development of the fly and leg regeneration of the cricket. The Ds-Ft signaling pathway is linked to the Warts-Hippo (Wts-Hpo) signaling pathway, leading to cell proliferation. Several lines of evidence have suggested that the Wts-Hpo signaling pathway is involved in the control of organ size, and that this pathway is regulated by Ds-Ft and Merlin-Expanded, which are linked to morphogens such as decapentaplegic/bone morphogenic protein, Wingless/Wnt, and epidermal growth factor. Here we review recent progress in elucidating mechanisms controlling leg size and shape in insects and vertebrates, focusing on the Ds-Ft signaling pathway. We also introduce a working model, Ds-Ft steepness model, to explain how steepness of the Ds-Ft gradient controls leg size along the proximodistal axis.
Tetsuya Bando, Yoshimasa Hamada, Kazuki Kurita, Taro Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Lowfat, a mammalian Lix1 homologue, regulates leg size and growth under the Dachsous/Fat signaling pathway during tissue regeneration., Developmental Dynamics, Vol.240, No.6, 1440-1453, 2011.
(Summary)
In the cricket Gryllus bimaculatus, missing distal parts of amputated legs are regenerated from blastemas based on positional information. The Dachsous/Fat (Ds/Ft) signaling pathway regulates blastema cell proliferation and positional information along the longitudinal axis during leg regeneration. Herein, we show that the Gryllus homologue of Lowfat (Gb'Lft), which modulates Ds/Ft signaling in Drosophila, is involved in leg regeneration. Gb'lft is expressed in regenerating legs, and RNAi against Gb'lft (Gb'lft(RNAi)) suppressed blastema cell hyperproliferation caused by Gb'ft(RNAi) or Gb'ds(RNAi) but enhanced that caused by Gb'kibra(RNAi) or Gb'warts(RNAi). In Gb'lft(RNAi) nymphs, missing parts of amputated legs were regenerated, but the length of the regenerated legs was shortened depending on the position of the amputation. Both normal and reversed intercalary regeneration occurred in Gb'lft(RNAi) nymphs, suggesting that Gb'Lft is involved in blastema cell proliferation and longitudinal leg regeneration under the Ds/Ft signaling pathway, but it is not required for intercalary regeneration.
Taro Mito, T Nakamura, T Band, Hideyo Ohuchi and Sumihare Noji : The advent of RNA interference in Entomology, Entomological Science, Vol.14, No.1, 1-8, 2011.
Taro Nakamura, Masato Yoshizaki, Syoutaro Ogawa, H Okamoto, Yohei Shinmyo, Tetsuya Bando, Hideyo Ohuchi, Sumihare Noji and Taro Mito : Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism, Current Biology, Vol.20, No.18, 1641-1647, 2010.
(Summary)
The mode of insect embryogenesis varies among species, reflecting adaptations to different life history strategies [1, 2]. In holometabolous insects, which include the model systems, such as the fruit fly and the red flour beetle, a large proportion of the blastoderm produces an embryo, whereas hemimetabolous embryos generally arise from a small region of the blastoderm [3]. Despite their importance in evolutionary studies, information of early developmental dynamics of hemimetabolous insects remains limited. Here, to clarify how maternal and gap gene products act in patterning the embryo of basal hemimetabolous insects, we analyzed the dynamic segmentation process in transgenic embryos of an intermediate-germ insect species, the cricket, Gryllus bimaculatus. Our data based on live imaging of fluorescently labeled embryonic cells and nuclei suggest that the positional specification of the cellular blastoderm may be established in the syncytium, where maternally derived gradients could act fundamentally in a way that is similar to that of Drosophila, namely throughout the egg. Then, the blastoderm cells move dynamically, retaining their positional information to form the posteriorly localized germ anlage. Furthermore, we find that the anterior head region of the cricket embryo is specified by orthodenticle in a cellular environment earlier than the gnathal and thoracic regions. Our findings imply that the syncytial mode of the early segmentation in long-germ insects evolved from a dynamic syncytial-to-cellular mode found in the present study, accompanied by a heterochronic shift of gap gene action.
Taro Mito, Taro Nakamura and Sumihare Noji : Evolution of insect development: to the hemimetabolous paradigm., Current Opinion in Genetics & Development, Vol.20, No.4, 355-361, 2010.
(Summary)
Mechanisms of insect development have been extensively studied in Drosophila melanogaster, a holometabolous insect. However, recent studies on other insects have gradually revealed that there exist new developmental paradigms. In this review, we focus on the new hemimetabolous paradigm. We highlight how hemimetabolous short-germ or intermediate-germ embryos establish the anterior/posterior (A/P) pattern and the importance of dynamic cell movement during germband formation. In hemimetabolous insects, orthodenticle, encoding a homeodomain-containing transcription factor, and wingless/Wnt signaling could play crucial roles in the A/P pattern formation. We also discuss recent evidence suggesting that insect developmental modes may have evolved by heterochronic shifts, while retaining certain universal metazoan features.
A Hamada, K Miyawaki, E Honda-sumi, K Tomioka, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Loss-of-function analyses of the fragile X-related and dopamine receptor genes by RNA interference in the cricket Gryllus bimaculatus, Developmental Dynamics, Vol.238, No.8, 2025-2033, 2009.
(Summary)
In order to explore a possibility that the cricket Gryllus bimaculatus would be a useful model to unveil molecular mechanisms of human diseases, we performed loss-of-function analyses of Gryllus genes homologous to human genes that are responsible for human disorders, fragile X mental retardation 1 (fmr1) and Dopamine receptor (DopR). We cloned cDNAs of their Gryllus homologues, Gb'fmr1, Gb'DopRI, and Gb'DopRII, and analyzed their functions with use of nymphal RNA interference (RNAi). For Gb'fmr1, three major phenotypes were observed: (1) abnormal wing postures, (2) abnormal calling song, and (3) loss of the circadian locomotor rhythm, while for Gb'DopRI, defects of wing posture and morphology were found. These results indicate that the cricket has the potential to become a novel model system to explore human neuronal pathogenic mechanisms and to screen therapeutic drugs by RNAi.
T Bando, Taro Mito, Y Maeda, T Nakamura, F Ito, T Watanabe, Hideyo Ohuchi and Sumihare Noji : Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration, Development, Vol.136, No.13, 2235-2245, 2009.
(Summary)
An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg.
(Keyword)
Animals / Cadherins / Cell Adhesion Molecules / Extremities / Gryllidae / In Situ Hybridization / Insect Proteins / Molecular Sequence Data / Neurofibromin 2 / Regeneration / Signal Transduction
T Takahashi, A Hamada, K Miyawaki, Y Matsumoto, Taro Mito, Sumihare Noji and M Mizunami : Systemic RNA interference for the study of learning and memory in an insect, Journal of Neuroscience Methods, Vol.179, No.1, 9-15, 2009.
(Summary)
RNA interference (RNAi) is a powerful technique for the study of molecular mechanisms underlying many biological processes, including brain functions. Among methods for RNAi, systemic administration of double-stranded RNA (systemic RNAi) is the most convenient for basic research as well as medical application, but it has yielded only limited success. To our knowledge, systemic RNAi has not been achieved for the study of learning and memory in any animals. Here we demonstrate successful systemic RNAi of the NOS gene coding for nitric oxide synthase, which, as we previously suggested, plays a critical role in the formation of olfactory long-term memory (LTM), in the nymphal cricket Gryllus bimaculatus. In situ hybridization demonstrated a high level of expression of NOS in a subset of Kenyon cells of the mushroom body, which is known to participate in olfactory learning and memory, in addition to some neurons around the antenna lobe and the base of the optic lobe. Injection of NOS double-stranded RNA (dsRNA) into the haemolymph completely impaired 1-day memory retention, although 30 min retention was unaffected. This impairment was fully rescued by injection of an NO donor, NOR3, thus suggesting that the effect of NOS dsRNA is through inhibition of NOS. Inhibition of NOS had no effects on recall of LTM. The results demonstrate that silencing of NOS expression by systemic RNAi impairs LTM formation. Systemic RNAi will become a useful method for study of the molecular mechanisms of learning and memory.
A Urasaki, Taro Mito, Sumihare Noji, R Ueda and K Kawakami : Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster, Gene, Vol.425, No.1-2, 64-68, 2008.
(Summary)
The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila.
(Keyword)
Animals / Cloning, Molecular / DNA Transposable Elements / Drosophila melanogaster / Gene Transfer Techniques / Oryzias / Transfection
T Nakamura, Taro Mito, K Miyawaki, Hideyo Ohuchi and Sumihare Noji : EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph, Developmental Biology, Vol.319, No.1, 46-55, 2008.
(Summary)
Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.
Taro Mito, Taro Nakamura, Isao Sarashina, CC Chang, S Ogawa, Hideyo Ohuchi and Sumihare Noji : Dynamic expression patterns of vasa during embryogenesis in the cricket Gryllus bimaculatus, Development Genes and Evolution, Vol.218, No.7, 381-387, 2008.
(Summary)
The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.
Taro Mito, Monica Ronco, Tomohiro Uda, Taro Nakamura, Hideyo Ohuchi and Sumihare Noji : Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus., Developmental Biology, Vol.313, No.1, 67-79, 2008.
(Summary)
The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb'exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb'exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb'exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb'exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb'exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.
Monica Ronco, Tomohiro Uda, Taro Mito, Alessandro Minelli, Sumihare Noji and Martin Klingler : Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus., Developmental Biology, Vol.313, No.1, 80-92, 2008.
(Summary)
Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb'hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb'wingless, Gb'dachshund, Gb'aristaless and Gb'Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb'hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb'hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb'hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.
T Nakamura, Taro Mito, T Bando, Hideyo Ohuchi and Sumihare Noji : Dissecting insect leg regeneration through RNA interference., Cellular and Molecular Life Sciences, Vol.65, No.1, 64-72, 2008.
(Summary)
Nymphs of hemimetabolous insects such as cockroaches and crickets exhibit a remarkable capacity for regenerating complex structures from damaged legs. Until recent years, however, approaches to elucidate the molecular mechanisms underlying the leg regeneration process have been lacking. Taking the cricket Gryllus bimaculatus as a model, we found that a phenotype related to regeneration frequently appears during leg regeneration, even though no phenotype is induced by RNA interference (RNAi) in the cricket nymph, designated as regeneration-dependent RNAi. Since then, we have investigated the functions of various genes encoding signaling factors and cellular adhesion proteins like Fat and Dachsous during leg regeneration. In this review, we summarize the classical knowledge about insect leg regeneration and introduce recent advances concerning the signaling cascades required for regenerating a leg. Our results provide clues to the mechanisms of regeneration which are relevant to vertebrate systems.
M Mitsumori, T Adachi, K Takayanagi, Taro Mito, Hideyo Ohuchi, S Kimura, M Kokubo, T Higuchi and Sumihare Noji : Film tomography as a tool for three-dimensional image construction and gene expression studies., Development Growth & Differentiation, Vol.49, No.7, 583-589, 2007.
(Summary)
In order to observe three-dimensional (3D) expression patterns of genes in whole animals, whole organs, or whole tissues, in situ hybridization (ISH) of many sections must be carried out and then used to construct a 3D image. For this purpose, we have developed an automatic microtome to prepare tissue sections with an adhesive film. We used commercially available film suitable for sectioning and ISH. We constructed a microtome and, after adherence of the film to a paraffin-embedded tissue block, cut the block with a blade to prepare sections on film. Then, the sections-on-film were automatically set in a plastic frame that was the same size as a conventional glass slide. With this automatic microtome, tissue sections can be made for ISH or immunohistochemistry in addition to conventional hematoxylin and eosin staining without specific training. We demonstrate that we can construct 3D images of gene expression patterns obtained by ISH on sections prepared with this automatic microtome. We have designated this method as 'Film Tomography (FITO)'.
(Keyword)
Animals / Gene Expression / In Situ Hybridization / Tomography
Taro Nakamura, Taro Mito, Yoshihisa Tanaka, Tetsuya Bando, Hideyo Ohuchi and Sumihare Noji : Involvement of the canonical Wnt/Wingless signaling in determination of the proximodistal positional values within the leg segment of the cricket Gryllus bimaculatus, Development Growth & Differentiation, Vol.49, No.2, 79-88, 2007.
Taro Mito and Sumihare Noji : Evolution of developmental systems underlying segmented body plans of bilaterian animals: insights from studies of segmentation in a cricket, Paleontological Research, Vol.10, No.4, 337-344, 2006.
(Summary)
Remarkable advances in developmental genetics in the past two decades allow us to approach the evolution of animal design by elucidating the molecular mechanisms underlying divergent body plans. The ancestry and evolution of segmented body plans in the bilaterians has been an active area of investigation in this field of study. Although segmentation mechanisms have been extensively studied for the fruit fly Drosophila melanogaster, Drosophila exhibits an evolutionarily derived mode of development, and molecular mechanisms underlying Drosophila segmentation may be unrepresentative for arthropods, even for insects. We have been studying the developmental system of the cricket, Gryllus bimaculatus, to understand more ancestral and general segmentation mechanisms for insects than those of Drosophila. In Gryllus, anterior segments are specified almost simultaneously, whereas posterior segments are specified sequentially in the extending posterior region. This mode of segmentation is general and probably ancestral for arthropods. Our RNA interference-based analyses of the functions and regulatory interactions of Gryllus orthologues of Drosophila segmentation genes have revealed surprisingly divergent aspects of the segmentation system in Gryllus in comparison with that of Drosophila. For example, the anteroposterior patterning in Gryllus is principally controlled by the caudal (cad) gene, probably without bicoid, unlike Drosophila. Comparisons of regulatory networks of segmentation genes between Gryllus and Drosophila suggest that regulatory interactions between the genes vary among insects, despite conservation of the network component genes. This implies that the molecular mechanisms of segmentation have changed dynamically during insect evolution, whereas the segmented body plan itself has been conserved. We also discuss evolution of developmental systems generating segment patterns in non-arthropod bilaterian animals.
Yohei Shinmyo, Taro Mito, T Uda, Taro Nakamura, Katsuyuki Miyawaki, Hideyo Ohuchi and Sumihare Noji : brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus., Development, Vol.133, No.22, 4539-4547, 2006.
(Summary)
In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Body Patterning / Cloning, Molecular / DNA Primers / DNA-Binding Proteins / Drosophila Proteins / Gryllidae / In Situ Hybridization / Lower Gastrointestinal Tract / Molecular Sequence Data / Morphogenesis / RNA Interference / Sequence Alignment / Sequence Analysis, DNA / Trans-Activators
A Yoshimura, A Nakata, Taro Mito and Sumihare Noji : The characteristics of karyotype and telomeric satellite DNA sequences in the cricket, Gryllus bimaculatus (Orthoptera, Gryllidae), Cytogenetic and Genome Research, Vol.112, No.3-4, 329-336, 2006.
(Summary)
The chromosomes derived from the Japanese population of Gryllus bimaculatus were characterized by C-banding and Ag-NOR staining. The chromosome number, 2n = 28 + XX (female)/XO (male), corresponded with that of other populations of G. bimaculatus, but the chromosome configuration in idiograms varied between the populations. NORs were carried on one pair of autosomes and appeared polymorphous. The positive C-bands located at the centromere of all chromosomes and the distal regions of many chromosome pairs, and the size and the distribution pattern of the distal C-heterochromatin showed differences among the chromosomes. In addition, this paper reports on the characteristics of HindIII satellite DNA isolated from the genome of G. bimaculatus. The HindIII repetitive fragments were about 0.54 kb long, and localized at the distal C-bands of the autosomes and the interstitial C-bands of the X chromosome. Molecular analysis showed two distinct satellite DNA sequences, named the GBH535 and GBH542 families, with high AT contents of about 67 and 66%, respectively. The two repetitive families seem to be derived from a common ancestral sequence, and both families possessed the same 13-bp palindrome sequence. The results of Southern blot hybridization suggest that the sequence of the GBH535 family is conserved in the genomic DNAs of Gryllus species, whereas the GBH542 family is a species-specific sequence.
(Keyword)
Animals / Antigens, Nuclear / Chromosome Banding / Chromosome Mapping / DNA, Satellite / Female / Gryllidae / Japan / Karyotyping / Male / Nuclear Proteins / Telomere / X Chromosome / Y Chromosome
Isao Sarashina, Taro Mito, Michiko Saito, Hiroyuki Uneme, Katsuyuki Miyawaki, Yohei Shinmyo, Hideyo Ohuchi and Sumihare Noji : Location of micropyles and early embryonic development of the two-spotted cricket Gryllus bimaculatus (Insecta, Orthoptera), Development Growth & Differentiation, Vol.47, No.2, 99-108, 2005.
(Summary)
Early embryogenesis of the two-spotted cricket Gryllus bimaculatus was examined by scanning electron microscopy and several fluorescence staining methods, with special reference to these four issues: (i) the location of micropyles; (ii) the transfer of the female pronucleus following meiosis; (iii) the timing of cellularization; and (iv) the process of the germ primordium formation. Between two and four micropyles lie in the mid-ventral region of the egg. The egg nucleus is at the mid-dorsal periphery of the new laid egg, and meiosis resumes and is completed there. The female pronucleus moves to the mid-ventral side, and fertilization occurs there. Energid starts to proliferate and migrates to the periphery of the egg, initiating blastoderm formation. Actin caps surround each superficial nucleus. Cellularization occurs during the blastoderm stage. At a late blastoderm stage, nuclei aggregate in both the posterolateral patch-like regions of the egg to form a germ primordium. The germ primordium looks like a pair of dumbbells. Both the patches shift towards the ventral side and fuse into a germ primordium. The germ primordium contracts to produce a clearly delineated germ band. Observations on distribution patterns of F-actin indicate that, all through the process, the germ primordium retains that unity, and is not separated into two parts.
Yohei Shinmyo, Taro Mito, Taro Matsushita, Isao Sarashina, Katsuyuki Miyawaki, Hideyo Ohuchi and Sumihare Noji : caudal is required for gnathal and thoracic patterning and for posterior elongation in the intermediate-germband cricket Gryllus bimaculatus, Mechanisms of Development, Vol.122, No.2, 231-239, 2005.
(Summary)
Although the molecular mechanisms directing anteroposterior patterning of the Drosophila embryo (long-germband mode) are well understood, how these mechanisms were evolved from an ancestral mode of insect embryogenesis remains largely unknown. In order to gain insight into mechanisms of evolution in insect embryogenesis, we have examined the expression and function of the orthologue of Drosophila caudal (cad) in the intermediate-germband cricket Gryllus bimaculatus. We observed that a posterior (high) to anterior (low) gradient in the levels of Gryllus bimaculatus cad (Gb' cad) transcript was formed in the early-stage embryo, and then Gb' cad was expressed in the posterior growth zone until the posterior segmentation was completed. Reduction of Gb' cad expression level by RNA interference resulted in deletion of the gnathum, thorax, and abdomen in embryos, remaining only anterior head. We found that the gnathal and thoracic segments are formed by Gb' cad probably through the transcriptional regulation of gap genes including Gb' hunchback and Gb' Kruppel. Furthermore, Gb' cad was found to be involved in the posterior elongation, acting as a downstream gene in the Wingless/Armadillo signalling pathways. These findings indicate that Gb' cad does not function as it does in Drosophila, suggesting that regulatory and functional changes of cad occurred during insect evolution. Since Wnt/Cdx pathways are involved in the posterior patterning of vertebrates, such mechanisms may be conserved in animals that undergo sequential segmentation from the posterior growth zone.
Katsuyuki Miyawaki, Taro Mito, Isao Sarashina, Hongjie Zhang, Yohei Shinmyo, Hideyo Ohuchi and Sumihare Noji : Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis, Mechanisms of Development, Vol.121, No.2, 119-130, 2004.
Hongjie Zhang, Yohei Shinmyo, Ayumi Hirose, Taro Mito, Yoshiko Inoue, Hideyo Ohuchi, Thanasis G. Loukeris, Paul Eggleston and Sumihare Noji : Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus, Development Growth & Differentiation, Vol.44, No.5, 409-417, 2002.
(Summary)
Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter-eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter-eGFP for the genetic transformation of this emerging model insect species.
(Keyword)
Actins / Amino Acid Sequence / Animals / Base Sequence / DNA Transposable Elements / Genes, Reporter / Genetic Vectors / Gryllidae / Molecular Sequence Data / Plasmids / Promoter Regions, Genetic
Taro Mito, Yoshiko Inoue, Shinsuke Kimura, Katsuyuki Miyawaki, Nao Niwa, Yohei Shinmyo, Hideyo Ohuchi and Sumihare Noji : Involvement of hedgehog, wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs, a verification of the modified boundary model, Mechanisms of Development, Vol.114, No.1, 27-35, 2002.
Yoshiko Inoue, Taro Mito, Katsuyuki Miyawaki, Kyoko Matsushima, Yohei Shinmyo, Tiffany A. Heanue, Graeme Mardon, Hideyo Ohuchi and Sumihare Noji : Correlation of expression patterns of homothorax, dachshund, and Distal-less with the proximodistal segmentation of the cricket leg bud, Mechanisms of Development, Vol.113, No.2, 141-148, 2002.
Yoshiko Inoue, Nao Niwa, Taro Mito, Hideyo Ohuchi, Hidefumi Yoshioka and Sumihare Noji : Expression patterns of hedgehog, wingless, and decapentaplegic during gut formation of Gryllus bimaculatus (cricket), Mechanisms of Development, Vol.110, No.1-2, 245-248, 2002.
Taro Mito and kazuyoshi Endo : PCR Survey of Hox Genes in the Crinoid and Ophiuroid: Evidence for Anterior Conservation and Posterior Expansion in the Echinoderm Hox Gene Cluster, Molecular Phylogenetics and Evolution, Vol.14, No.3, 375-388, 2000.
(Summary)
To help elucidate the cluster organization of Hox genes in echinoderms, we amplified a homeobox region by polymerase chain reaction (PCR) and cloned and sequenced the PCR products for the comatulid crinoid Oxycomanthus japonicus and the ophiuroid Stegophiura sladeni. The crinoid had at least three anterior, four medial, and four posterior genes, and the ophiuroid had at least one anterior, three medial, and six (one of which being a possible trans-paralog) posterior genes. The survey of the crinoid detected all three anterior complements (PG1-3 genes). It was inferred that the Hox genes of each species are organized into a single cluster and that a novel cognate group of echinoderm posterior genes (tentatively termed HboxP9) exists among echinoderms in addition to the known posterior genes Hbox4, Hbox7, and Hbox10. The results, combined with the data of other echinoderm classes, strongly suggest that the presence of a single Hox gene cluster is a common feature among echinoderms and that the cluster has the general features of the deuterostome Hox gene cluster, i.e., the anterior conservation and posterior expansion. The results of the ophiuroid imply that the posterior genes in this class diversified after the phylum Echinodermata had been established.
Taro Mito and Kazuyoshi Endo : A PCR Survey of Hox Genes in the Sea Star, Asterina minor, Molecular Phylogenetics and Evolution, Vol.8, No.2, 218-224, 1997.
(Summary)
The sea star, Asterina minor, was surveyed for Hox genes using the method of PCR and subsequent sequence determination. Seven different Hox-type homeobox fragments and homeobox fragments of two other types, the Gbx-type and the Xlox-type, were identified. The results of comparative analysis with known homeobox sequences suggest that the sea star has only one Hox gene cluster including two genes of the anterior group, four genes of the medial group, and one gene of the posterior group. The existence of a gene of the cognate group 1 has not been known in echinoderm species. Each of the other fragments indicated a definite relationship with one of sea urchin homeoboxes. The hypothetical cluster in the sea star is consistent with the results published for another class of echinoderm, sea urchins, in the putative number of cluster. The present result provides strong evidence that a single Hox cluster is common to echinoderms and its structure in the anterior region is more similar to other deuterostomes than previously thought.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / DNA Primers / molecular evolution / Genes, Homeobox / Molecular Sequence Data / Multigene Family / Polymerase Chain Reaction / Sea Urchins / Sequence Homology, Amino Acid / Starfish
Taro Mito, Yoshiyasu Ishimaru, Takahito Watanabe, Taro Nakamura, Guillem Ylla, Sumihare Noji and G Cassandra Extavour : Cricket: The third domesticated insect., Current Topics in Developmental Biology, Vol.147, 291-306, Mar. 2022.
(Summary)
Many researchers are using crickets to conduct research on various topics related to development and regeneration in addition to brain function, behavior, and biological clocks, using advanced functional and perturbational technologies such as genome editing. Recently, crickets have also been attracting attention as a food source for the next generation of humans. In addition, crickets are increasingly being used as disease models and biological factories for pharmaceuticals. Cricket research has thus evolved over the last century from use primarily in highly important basic research, to use in a variety of applications and practical uses. These insects are now a state-of-the-art model animal that can be obtained and maintained in large quantities at low cost. We therefore suggest that crickets are useful as a third domesticated insect for scientific research, after honeybees and silkworms, contributing to the achievement of global sustainable development goals.
Tetsuya Bando, Taro Mito, Yoshimasa Hamada, Yoshiyasu Ishimaru, Sumihare Noji and Hideyo Ohuchi : Molecular mechanisms of limb regeneration: insights from regenerating legs of the cricket Gryllus bimaculatus, The International Journal of Developmental Biology, Vol.62, No.6-7-8, 559-569, Jun. 2018.
(Summary)
This review summarizes recent advances in leg regeneration research, focusing on the cricket Gryllus bimaculatus. Recent studies have revealed molecular mechanisms on blastema formation, establishment of positional information, and epigenetic regulation during leg regeneration. Especially, these studies have provided molecular bases in classical conceptual models such as the polar coordinate model, the intercalation model, the boundary model, the steepness model, etc., which were proposed to interpret regeneration processes of the cockroach legs. When a leg is amputated, a blastema is formed through the activation of the Janus-kinase (Jak)/Signal-Transduction-and-Activator-of-Transcription (STAT) pathway. Subsequently, the Hedgehog/Wingless/Decapentaplegic/Epidermal-growth-factor pathways instruct distalization in the blastema, designated as the molecular boundary model. Downstream targets of this pathway are transcription factors Distal-less (Dll) and dachshund (dac), functioning as key regulators of proximodistal pattern formation. Dll and dac specify the distal and proximal regions in the blastema, respectively, through the regulation of tarsal patterning genes. The expression of leg patterning genes during regeneration may be epigenetically controlled by histone H3K27 methylation via Enhancer-of-zeste and Ubiquitously-transcribed-tetratricopeptide-repeat-gene-X-chromosome. For the molecular mechanism of intercalation of the missing structures between the amputated position and the most distal one, Dachsous/Fat (Ds/Ft) steepness model has been proposed, in which the Ds/Ft pathway maintains positional information and determines leg size through dac expression. This model was theoretically verified to interpret the experimental results obtained with cricket legs. Availability of whole-genome sequence information, regeneration-dependent RNA interference, and genome editing technique will have the cricket be an ideal model system to reveal gene functions in leg regeneration.
Takeshi Nikawa, Katsuyuki Miyawaki, Akira Takahashi, Takahito Watanabe and Taro Mito : ANTI-MUSCLE ATROPHIC PROTEIN FOOD SOURCE IN SPACE: DEVELOPMENT OF A RECIRCULATORY REARING SYSTEM FOR SOYBEANS AND CRICKETS, 45th COSPAR Scientific Assembly-COSPAR 2024, Jul. 2024.
2.
Shintaro Inoue, Takahito Watanabe, Hamaguchi Taiki, Fujie Kai, Shimamura Ayane, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji and Taro Mito : Artificial modification of cricket body color: breeding for the next-generation of protein supply, International Conference of Non-Traditional Arthropod Model Systems, Aug. 2023.
3.
Fujie Kai, Shintaro Inoue, Hamaguchi Taiki, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji, Takahito Watanabe and Taro Mito : The discovery of two paralogous dopamine-synthase genes in the two-spotted cricket Gryllus bimaculatus, International Conference of Non-Traditional Arthropod Model Systems, Aug. 2023.
4.
Taro Mito : Genome editing in the cricket Gryllus bimaculatus, 25th International Congress of Entomology, Vol.1630, 219-233, Orlando, Sep. 2016.
Matsuoka Yuji, Takahito Watanabe, Sayuri Tomonari, Sumihare Noji and Taro Mito : Functional analysis of a Hox gene, abdominal-A, using CRISPR/Cas9 system in the cricket Gryllus bimaculatus, International Tribolium Meeting 2015, Berkeley, USA, Aug. 2015.
6.
Takahito Watanabe, Matsuoka Yuji, Sayuri Tomonari, Kurita Chinami, Sumihare Noji and Taro Mito : Genome editing in the two-spotted cricket, Gryllus bimaculatus, using CRISPR/Cas9 system, Insect Genetic Technologies Workshop, Manhattan, Kansas, USA, Jun. 2015.
7.
Taro Mito, Itoh Takehiko, Morimoto Hiroya, Kajitani Ray, Toyoda Atsushi, Sayuri Tomonari, Fuketa Masao, Takahito Watanabe, Matsuoka Yuji and Sumihare Noji : Genome sequencing and annotation of the cricket Gryllus bimaculatus, a hemimetabolous insect model, Ninth Annual Arthropod Genomics Symposium, Manhattan, Kansas, USA, Jun. 2015.
8.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by TALEN and CRISPR/Cas systems, X meeting for Spanish Society for Developmental Biology (SEBD), Madrid, Oct. 2014.
9.
Taro Mito : Whole-genome sequencing and targeted genome editing in the cricket G. bimaculatus, Hokkaido Neuroethology Workshops 2014, 札幌, Jul. 2014.
10.
Sumihare Noji, Taro Mito, Bando Tetsuya, Nakamura Taro, Takahito Watanabe, Ishimaru Yoshiyasu and Hideyo Ohuchi : Regeneration of insect legs from stem cells, Thirteenth International Congress on Invertebrate Reproduction and Development, Detroit, Detroit, MI, USA, Jul. 2014.
11.
Nakamura Taro, Sumihare Noji and Taro Mito : Molecular mechanisms underlying early embryonic patterning and germ cell specification in the cricket, International Symposium on RNAi and Genome editing methods, Tokushima, Japan, Mar. 2014.
12.
Takahito Watanabe, Sumihare Noji and Taro Mito : Targeted genome modifications in the cricket, Gryllus bimaculatus, using CRISPR/Cas9 system, International Symposium on RNAi and Genome editing methods, Tokushima, Japan, Mar. 2014.
13.
Taro Mito : RNAi analysis and genome sequencing in the cricket Gryllus bimaculatus, a model for evolutionary developmental studies, International Symposium on RNAi and Genome editing methods, Tokushima, Japan, Mar. 2014.
14.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, H Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system., International Symposium on RNAi and Genome Editing Methods, Tokushima, Mar. 2014.
15.
Taro Mito, Takahito Watanabe and Sumihare Noji : Genome modification technology in the cricket Gryllus bimaculatus, 1st Asian Invertebrate Immunity Symposium, Busan, Feb. 2014.
16.
Takahito Watanabe, Matsuoka Yuji, Sumihare Noji and Taro Mito : Targeted genome editing in the cricket, Gryllus bimaculatus, using CRISPR/Cas9 system, FASEB SRC on Genome Engineering-Cutting-Edge Research and Applications, Nassau, Bahamas, Jan. 2014.
17.
Takahito Watanabe, Ochiai Hiroshi, Sakuma Tetsushi, Ishihara Satoshi, Nakamura Taro, Yamamoto Takashi, Sumihare Noji and Taro Mito : Targeted genome modifications in the cricket, Gryllus bimaculatus, Conference of Transposition & Genome Engineering 2013, Budapest, Hungary, Sep. 2013.
18.
Taro Mito : Gryllus bimaculatus -a hemimetabolous insect model for functional genomics, iBeetle symposium"New horizons in molecular Zoology"(Invited speaker), Göttingen, Germany, Mar. 2013.
19.
Taro Mito and T Watanabe : Genome modification in a hemimetabolous insect Gryllus bimaculatus, Janelia Workshop on Genomic Modification in Model and Non-Model Insects(Invited speaker), HHMI Janelia Farm Campus, Ashburn, USA, Mar. 2013.
20.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Hadley W. Horch, Naoya Hamaguchi, Taro Nakamura, Tetsuya Bando, Hideyo Ohuchi, Takashi Yamamoto, Sumihare Noji and Taro Mito : Gene knockout in a hemimetabolous insect Gryllus bimaculatus by nontransgenic genome modification with zinc-finger and TALE nucleases, Asia-Pacific Developmental Biology Conference, Taipei, Taiwan, Oct. 2012.
21.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Taro Nakamura, Taro Mito, Takashi Yamamoto and Sumihare Noji : Efficient production of knockout crickets using custom designed nucleases, ZFNs and TALENs, FASEB Science Research Conferences: Genome Engineering; Research & Applications, Lucca, Italy, Sep. 2012.
22.
Taro Mito, Nakamura Taro, Bando Tetsuya, Watanabe Takahito and Sumihare Noji : Exploring mechanisms of embryonic patterning in Gryllus bimaculatus, a hemimetablous insect model system, [Symposium: From embryo to metamorphosis: Genes for insect development (Organizers: Sumihare Noji and Martin Klingler)], 24th International Congress of Entomology, Daegu, Korea, Aug. 2012.
23.
Tetsuya Bando, Yoshimasa Hamada, Taro Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Dachsous/Fat signaling via Hippo/Salvador/Warts pathway regulates cell proliferation and pattern formation during leg regeneration in the cricket, 24th International Congress of Entomology, Daegu, Korea, Aug. 2012.
24.
Taro Nakamura, Taro Mito, Tetsuya Bando and Sumihare Noji : Role of Wnt and BMP signaling pathways in the regional specification of early blastoderm in the cricket Gryllus bimaculatus,, 24th International Congress of Entomology, Daegu, Korea, Aug. 2012.
25.
T Watanabe, H Ochiai, T Sakuma, T Nakamura, Taro Mito, Hideyo Ohuchi, T Yamamoto and Sumihare Noji : Efficient production of knockout crickets using zinc-finger nucleases, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
26.
T Nakamura, Taro Mito, T Bando and Sumihare Noji : Involvement of Wnt and BMP signaling pathways in the regional specification of early blastoderm in the cricket Gryllus bimaculatus, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
27.
T Bando, Y Matsuoka, Y Hamada, T Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Molecular mechanisms underlying cell proliferation and pattern formation during leg regeneration in Gryllus bimaculatus, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
28.
Y Matsuoka, T Bando, T Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Functional analysis of epigenetic regulation during embryogenesis of the cricket, Gryllus bimaculatus, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
29.
Makoto Mizunami, T Takahashi, A Hamada, Katsuyuki Miyawaki, Y Matsumoto, Taro Mito and Sumihare Noji : Systemic RNA interference for the study of long-term memory formation in the cricket, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
30.
Taro Mito, T Nakamura, T Watanabe, H Ochiai, T Sakuma, T Bando, Hideyo Ohuchi and Sumihare Noji : Exploring mechanisms of embryonic patterning in Gryllus bimaculatus, a hemimetabolous insect model system, The 2nd International Conference on the Cricket / RNAi Symposium, tokushima,Japan, Mar. 2012.
31.
Taro Mito, T Nakamura, T Watanabe, T Band and Sumihare Noji : Functional genomics of the cricket Gryllus bimaculatus, a model system for regeneration and evolutionary developmental studies, 5th Annual Arthropod Genomics Symposium, Kansas City,USA, Jun. 2011.
32.
Taro Mito, T Nakamura, T Bando and Sumihare Noji : Ancestral developmental mechanisms in insects revealed by RNAi analysis of cricket genes, [Symposium: RNA interference- comparative studies of gene functions in invertebrates, 8th International Congress on Comparative Physiology and Biochemistry, Nagoya, Jun. 2011.
33.
T Nakamura, Taro Mito, M Yoshizaki, T Bando and Sumihare Noji : Dynamic control of positional specification in a primitive mode of insect segmentation, フランス(パリ第7大学), Jul. 2010.
34.
Sumihare Noji, Tetsuya Bando, Taro Mito, Taro Nakamura, Takahito Watanabe and Hideyo Ohuchi : Regulation of leg size and shape by the Dachsous/Fat signalling pathway during cricket leg regeneration, Washington, DC, USA, Apr. 2010.
35.
Taro Mito, Taro Nakamura, Masato Yoshizaki, Tetsuya Bando, Hideyo Ohuchi and Sumihare Noji : Highly dynamic cell behavior during early development in the intermediate germ insect Gryllus bimaculatus, as revealed by analyses of transgenic embryos, Washington DC, USA, Apr. 2010.
36.
Taro Mito, Katsuyuki Miyawaki, Y Shinmyo, Hideyo Ohuchi and Sumihare Noji : Genome-wide RNAi in the cricket Gryllus bimaculactus as a new model system to study gene functions, 10th Anniversary of Kazusa DNA Research Institute 14th International Workshop, Japan, Oct. 2004.
Sumihare Noji, Taro Mito, Y Tanaka, Katsuyuki Miyawaki, (名) Shinmyo and Hideyo Ohuchi : Involvement of Wnt and EGF signaling systems in regeneraion of cricket legs, 8th international Conference Limb Development and Regeneration, 47, Dundee, UK, Jul. 2004.
38.
Sumihare Noji, Yoshiko Inoue, Taro Mito and Hideyo Ohuchi : Limb development and regeneration of the cricket leg: Expression patterns of hedgehog, wingless and decepentaplegic, 7th International conference on limb development and regeneration, Vol.90, No.1, 20, Aussois, France, Aug. 2000.
39.
Sumihare Noji, Y Inoue, Taro Mito and Hideyo Ohuchi : Limb development and Regeneration of the cricket leg: Expression patterns of hedgehog, wingless and decapentaplegic, 7th International Conference on Limb Development and Regeneration, France, May 2000.
40.
Keiichi Takahashi, Taro Mito, H Imai and M Fujii : Bi-directional transport of fluid and particles on uniformly ciliated epithelium, Comparative Biochemistry and Physiology, Vol.124A, No.Supplement 1, 83, Calgary, Aug. 1999.
41.
Tatsuo Oji and Taro Mito : Skeletal development in Oxycomanthus japonica and its paleontologic implications, The 5th European Conference on Echinoderms, 74, Milan, Sep. 1998.
42.
Taro Mito and Kazuyoshi Endo : Conservation of the anterior region of Hox gene cluster in echinoderms, The 5th European Conference on Echinoderms, 53, Milan, Sep. 1998.
Hamaguchi Taiki, Shintaro Inoue, Takahito Watanabe, Yoshiyasu Ishimaru and Taro Mito : フタホシコオロギにおけるクチクラ形成と色素合成に関わる遺伝子の機能解析, The 44th Annual Meeting of the Molecular Biology Society of Japan, Dec. 2021.
19.
Ohde Takahiro, Taro Mito and Niimi Teruyuki : A hemimetabolous wing development implicates an essential step for novel insect wing evolution, The 43rd Annual Meeting of the Molecular Biology Society of Japan, Dec. 2020.
20.
Higashihara Aya, Yoshiyasu Ishimaru, Matsumura Saki, Kawamoto Kohei, Sayuri Tomonari, Sumihare Noji and Taro Mito : Gene knock-out analysis of a metamorphosis factor Myoglianin in the cricket Gryllus bimaculatus, The 43rd Annual Meeting of the Molecular Biology Society of Japan, Online, Dec. 2020.
Takuya Watari, Yoshiyasu Ishimaru, Sumihare Noji and Taro Mito : Involvement of macrophages in leg regeneration of the cricket Gryllus bimaculatus, 52nd Annual Meeting of the Japanese Society of Developmental Biologists, May 2019.
25.
Kohei Kawamoto, Mayuko Matsuda, Takahisa Yamashita, Takahito Watanabe, Sayuri Tomonari, Yoshiyasu Ishimaru, Sumihare Noji and Taro Mito : Precise in-frame integration of a GFP gene using microhomology-mediated knock-in technology in Gryllus bimaculatus, 52nd Annual Meeting of the Japanese Society of Developmental Biologists, May 2019.
26.
Takahisa Yamashita, Taro Mito, Yoshiyasu Ishimaru, Takahito Watanabe, Sayuri Tomonari, Kohei Kawamoto and Mayuko Matuda : Generation of an enhancer-trap strain of the scalloped gene in the cricket Gryllus bimaculatus, 52nd Annual Meeting of the Japanese Society of Developmental Biologists, May 2019.
27.
Yuki Nakamura, Sayuri Tomonari, Kohei Kawamoto, Takahito Watanabe, Yoshiyasu Ishimaru, Sumihare Noji and Taro Mito : Resolving the correlation between phenotype and genotype in a segmentation gene even-skipped in the cricket Gryllus bimaculatus, 52nd Annual Meeting of the Japanese Society of Developmental Biologists, May 2019.
28.
Taro Mito : フタホシコオロギを用いた研究におけるゲノム編集技術の活用, 第63回日本応用動物昆虫学会大会・小集会, Mar. 2019.
Taro Mito : ゲノム改変技術を用いたフタホシコオロギの発生機構の研究, 日本節足動物発生学会第53回大会, May 2017.
33.
Matsuda Mayuko, Matsuoka Yuji, Yoshiyasu Ishimaru, Sayuri Tomonari, Takahito Watanabe, Sumihare Noji and Taro Mito : Functional analysis of a Hox gene, abdominal-A, in the cricket Gryllus bimaculatus using a CRISPR/Cas9-mediated gene knock-in system, Japanese Society of Developmental Biologists, May 2017.
34.
Nakamura Yu-Ki, Kawamoto Kohei, Sayuri Tomonari, Matsuda Mayuko, Takahito Watanabe, Yoshiyasu Ishimaru, Uemura Natsuki, Sumihare Noji and Taro Mito : even-skipped is required for segmentation and elongation of embryos in the cricket Gryllus bimaculatusas revealed by CRISPR/Cas9-based gene knock-out., Japanese Society of Developmental Biologists, May 2017.
35.
Masamitsu Takai, Takahito Watanabe, Sayuri Tomonari and Taro Mito : フタホシコオロギの食用化に向けた生産システムの検討, 第61回日本応用動物昆虫学会大会, Mar. 2017.
Kohei Kawamoto, Sayuri Tomonari, Yuji Matsuoka, Takahito Watanabe, Yoshiyasu Ishimaru, Sumihare Noji and Taro Mito : even-skipped acts principally as a gap gene in the cricket Gryllus bimaculatus as revealed by CRISPR/Cas9-based gene knockout analysis, JSDB Special Symposium: Frontier of Developmental Biology Hosted by JSDB, Jun. 2016.
Silvia Mitsui, Akihiro Yasue, Issei Imoto, Seiichi Oyadomari, Sumihare Noji, Taro Mito and Eiji Tanaka : In vivo study of Msx1 gene in mice using CRISPR/Cas system, 第47回日本発生生物学会, May 2014.
47.
Yuji Matsuoka, Tetsuya Bando, Takahito Watanabe, Sumihare Noji and Taro Mito : Functions of Polycomb group gene in regulation of Hox gene expression in a primitive mode of insect embryogenesis in the cricket Gryllus bimaculatus, 第47回日本発生生物学会, May 2014.
48.
Yoshimasa Hamada, Tetsuya Bando, Taro Mito, Kenji Tomioka, Sumihare Noji and Hideyo Ohuchi : Epigenetic regulation of gene expressions via methylation on histone H3 27th lysine residue during leg regeneration, 第47回日本発生生物学会, May 2014.
49.
Takahito Watanabe, Yuji Matsuoka, Taro Mito and Sumihare Noji : Targeted gene disruption in the cricket, Gryllus bimaculatus, using CRISPR/Cas9 system, 第47回日本発生生物学会, May 2014.
50.
Taro Mito, 渡辺 崇人 and Sumihare Noji : CRISPR/Casシステムを用いたフタホシコオロギにおけるゲノム編集, 第58回日本応用動物昆虫学会大会, Mar. 2014.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system, 第36回日本分子生物学会, Dec. 2013.
53.
Akihiro Yasue, Silvia Naomi Mitsui Akagi, Teppei Watanabe, T Sakuma, Seiichi Oyadomari, T Yamamoto, Sumihare Noji, Taro Mito and Eiji Tanaka : A high efficient gene targeting in one-cell mouse embryos mediated by TALEN and CRISPR/Cas system, 第36回日本分子生物学会, Dec. 2013.
渡辺 崇人, 松岡 佑児, 石原 聡, 山本 卓, Sumihare Noji and Taro Mito : ゲノム編集技術によるノックアウトコオロギの作製, 第15回日本進化学会大会, Aug. 2013.
58.
Nakamura Taro, Sumihare Noji and Taro Mito : Regulation of Wnt and BMP signaling pathways in the regional specification of early blastoderm in the cricket Gryllus bimaculatus, 第46回日本発生生物学会年会, May 2013.
59.
Bando Tetsuya, Taro Mito, Ohuchi Hideyo and Sumihare Noji : JAK/STAT signaling promotes blastemal cell proliferation during leg regeneration in the cricket Gryllus bimaculatus, 第46回日本発生生物学会年会, May 2013.
60.
松岡 佑児, 板東 哲哉, 中村 太郎, 渡辺 崇人, Sumihare Noji and Taro Mito : Epigenetic regulation of Hox gene expression by PcG genes in a primitive mode of insect embryogenesis in the cricket Gryllus bimaculatus, 第46回日本発生生物学会年会, May 2013.
61.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Taro Nakamura, Taro Mito, Takashi Yamamoto and Sumihare Noji : Targeted genome modifications using ZFNs and TALENs in the cricket Gryllus bimaculatus, 第46回日本発生生物学会年会, May 2013.
62.
T Bando, Y Ishimaru, T Kida, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Molecular mechanisim of regulation of blastemal cell proliferation during leg regeneration in Gryllus bimaculatus, 第35回日本分子生物学会年会, Dec. 2012.
63.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Taro Nakamura, Taro Mito, Takashi Yamamoto and Sumihare Noji : Generation of knockout crickets using ZFNs and TALENs, 第35回日本分子生物学会年会, Dec. 2012.
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Taro Nakamura, Taro Mito, Hideyo Ohuchi, Takashi Yamamoto and Sumihare Noji : Efficient production of knockout crickets using zinc-finger nucleases, 第45回日本発生生物学会年会, May 2012.
66.
Tetsuya Bando, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Angiomotin regulates cell proliferation cooperatively with Expanded and Merlin during leg regeneration in Gryllus bimaculatus, 第45回日本発生生物学会年会, May 2012.
67.
Taro Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Regulation of orthodenticle and Wnt/Cad signaling pathway in anterior-posterior axis patterning during cricket early embryogenesis, 第45回日本発生生物学会年会, May 2012.
68.
Sumihare Noji, T Bando, Taro Mito and Hideyo Ohuchi : Molecular mechanisms underlying insect leg regeneration: from wound healing to leg size determination, 第34回日本分子生物学会年会, 2011.
69.
Y Matsuoka, T Bando, T Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Polycomb group genes epigenetically determines segmental identity in the cricket, Gryllus bimaculatus, 第34回日本分子生物学会年会, Dec. 2011.
70.
T Bando, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Angiomotin regulates leg size cooperatively with Expanded and Merlin during regeneration in Gryllus bimaculatus, 第34回日本分子生物学会年会, Dec. 2011.
71.
T Watanabe, H Ochiai, T Sakuma, T Nakamura, Taro Mito, Hideyo Ohuchi, T Yamamoto and Sumihare Noji : Making knockout crickets with zinc-finger nucleases, 第34回日本分子生物学会年会, Dec. 2011.
72.
Taro Mito, T Nakamura, T Watanabe, H Ochiai, T Sakuma, T Bando, Hideyo Ohuchi and Sumihare Noji : Exploring molecular mechanisms of early embryogenesis in the cricket Gryllus bimaculatus, 第34回日本分子生物学会年会, Dec. 2011.
73.
A Nakai, M Yoshizaki, Taro Mito, T Nakamura, T Bando, Hideyo Ohuchi and Sumihare Noji : Role of the orthodenticle gene in an ancestral mode of insect embryogenesis, as revealed by expression and functional analyses in the cricket Gryllus bimaculatus, 第44回日本発生生物学会年会, May 2011.
74.
T Nakamura, Taro Mito, M Yoshizaki, A Nakai, Hideyo Ohuchi and Sumihare Noji : Imaging of transgenic cricket embryos reveals cell movements consist with a syncytial patterning mechanism, 第44回日本発生生物学会年会, May 2011.
75.
Y Hamada, T Bando, Y Matsuoka, T Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Epigenetic regulation of gene expressions during leg regeneration in the two-spotted cricket, Gryllus bimaculatus, 第44回日本発生生物学会年会, May 2011.
76.
T Bando, Y Hamada, T Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Regulatory mechanism of blastemal cells mediated by polarity complexes via Dachsous/Fat and Hippo/Salvador/Warts pathway during leg regeneration in Gryllus bimaculatus, 第44回日本発生生物学会年会, May 2011.
77.
T Watanabe, H Ochiai, T Sakuma, M Asahina, T Nakamura, Taro Mito, Hideyo Ohuchi, T Yamamoto and Sumihare Noji : Targeted manipulation of genes with zinc finger nucleases in the cricket, Gryllus bimaculatus, 第44回日本発生生物学会年会, May 2011.
78.
K Matsuda, T Nakamura, T Bando, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Spatio-temporally controlled misexpression of genes using the GAL4/UAS system in the cricket, Gryllus bimaculatus, 第44回日本発生生物学会年会, May 2011.
Takahito Watanabe, H Ochiai, T Sakuma, Miki Asahina, Taro Nakamura, Taro Mito, Hideyo Ohuchi, T Yamamoto and Sumihare Noji : Targeted manipulation of genes with zinc finger nucleases in the cricket, Gryllus bimaculatus, 第33回日本分子生物学会年会, Dec. 2010.
81.
Kazuki Kurita, Akira Takagi, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Functions of the Drosophila retinal determination gene homologoues in eye development of a Hemimetabolous insect Gryllus bimaculatus, 第33回日本分子生物学会年会, Dec. 2010.
82.
Tetsuya Bando, Yuji Matsuoka, Yoshimasa Hamada, Taro Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Epigenetic regulation of gene expressions during leg regeneration in the two-spotted cricket Gryllus bimaculatus, 第33回日本分子生物学会年会, Dec. 2010.
83.
Masato Yoshizaki, Taro Nakamura, Taro Mito, Tetsuya Bando, Takahito Watanabe, Hideyo Ohuchi and Sumihare Noji : Functions of the orthodenticle-related genes during embryogenesis in the cricket Gryllus bimaculatus, 第33回日本分子生物学会年会, Dec. 2010.
Koji Matsuda, Taro Nakamura, F Ito, Taro Mito, Tetsuya Bando and Sumihare Noji : Development of enhancer trap lines using the GAL4/UAS system in the cricket, Gryllus bimaculatus, 第43回日本発生生物学会年会, Jun. 2010.
87.
Noha Dabour, Tetsuy Bando, Taro Nakamura, Katsuyuki Miyawaki, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Control of body size by chico and epidermal growth factor receptor, as revealed by systemic nymphal RNA interference in the cricket, 第43回日本発生生物学会年会, Jun. 2010.
88.
Yuji Matsuoka, Tetsuya Bando, Taro Nakamura, Hideyo Ohuchi, Sumihare Noji and Taro Mito : Enhancer of zeste epigenetically regulates leg development in the cricket Gryllus bimaculatus, 第43回日本発生生物学会年会, Jun. 2010.
89.
T Nakamura, Taro Mito, T Bando, Hideyo Ohuchi and Sumihare Noji : Dynamic control of positional specification in a primitive mode of insect segmentation, 第43回日本発生生物学会年会, Jun. 2010.
90.
Kazuki Kurita, Yohei Shinmyo, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Divergent function of Delta/Notch signaling in formation of body segments in the intermediate-germband cricket Gryllus bimaculatus, 第43回日本発生生物学会年会, Jun. 2010.
91.
Tetsuya Bando, Taro Nakamura, Taro Mito, Hideyo Ohuchi and Sumihare Noji : Lowfat regulates leg size and growth under the Dachsous/Fat signaling during regeneration in Gryllus bimaculatus, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jun. 2010.
Development and evolution of heteromorphosis in insects (Project/Area Number: 19H02970 )
Study on regulatory mechanisms of postembryonic development in a model system of hemimetabolous insects (Project/Area Number: 17H03945 )
Identification of key developmental changes for insect wing evolution (Project/Area Number: 16K18825 )
Fundamental study of safe treatment for overcoming muscle atrophy condition with aging (Project/Area Number: 16K13045 )
Epigenetic regulation for blastema formation during leg regeneration in the cricket (Project/Area Number: 15K06897 )
Study on genomic basis of morphogenesis in hemimetabolous insects using novel genetic modification techniques (Project/Area Number: 26292176 )
Evolutionary developmental study by using genome-editing technologies for in vivo reconstruction of gene networks (Project/Area Number: 25650080 )
Mechanisms for generating positional information and cell movements during early embryogenesis in the hemimetabolous insect (a cricket) (Project/Area Number: 23687033 )
Mechanisms of blastema formation after leg amputationin Gryllus bimaculatus (Project/Area Number: 22370080 )
Mechanisms underlying determination of positional information through Dachsous/Fat signaling system (Project/Area Number: 22124003 )
Development of novel efficient techniques for genetic manipulation and their application to explore the mechanisms of body axis determination in the cricket Gryllus bimaculatus (Project/Area Number: 21770237 )
Functional evolution of the gap gene group in early embryogenesis of the insect (Project/Area Number: 19370095 )
Identification of organ-specific cis-elements for the Fgf10 gene and their utilization for developmental-engineering tools (Project/Area Number: 16570177 )