研究者詳細 - 堀田　耕司

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 μM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

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Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

Matsuo K, Tamura R, Hotta K, Okada M, Takeuchi A, Wu Y, Hashimoto K, Takano H, Momose A, Nishino A.

Front Cell Dev Biol. 9 2021年12月

研究論文（学術雑誌）, 査読有り

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Spatiotemporal dynamics of single cell stiffness in the early developing ascidian chordate embryo

Fujii Y., Koizumi W.C., Imai T., Yokobori M., Matsuo T., Oka K., Hotta K., Okajima T.

Communications Biology （Communications Biology） 4 （ 1 ） 2021年12月

During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.

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Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs)

Arimura Y., Shindo Y., Yamanaka R., Mochizuki M., Hotta K., Nakahara T., Ito E., Yoshioka T., Oka K.

PLoS ONE （PLoS ONE） 16 （ 5 May ） 2021年05月

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

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High responsiveness of auditory neurons to specific combination of acoustic features in female songbirds

Inda M., Hotta K., Oka K.

European Journal of Neuroscience （European Journal of Neuroscience） 53 （ 5 ） 1412 - 1427 2021年03月

ISSN 0953816X

Zebra finch (Taeniopygia guttata) is a songbird species in which males sing their unique songs to attract females who then select their preferred male. Acoustic features in the songs of individual males are important features for female auditory perception. While the male of this species is a classic model of vocal production, it has been little known about auditory processing in female. In the higher auditory brain regions, the caudomedial mesopallium (CMM) and nidopallium (NCM) contribute to female's sound recognition, we, therefore, extracted acoustic features that induce neural activities with high detection power on both regions in female finches. A multiple linear regression analysis revealed that neurons were sensitive to mean frequency and Wiener entropy. In addition, we performed an experiment with modified artificial songs and harmonic songs to directly investigate neural responsiveness for deriving further evidence for the contribution of these two acoustic features. Finally, we illustrated a specific ratio combining these two acoustic features that showed highest sensitivity to neural responsiveness, and we found that properties of sensitivity are different between CMM and NCM. Our results indicate that the mixture of the two acoustic features with the specific ratio is important in the higher auditory regions of female songbirds, and these two regions have differences in encoding for sensitivity to these acoustic features.

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Two-Round Ca 2+ transient in papillae by mechanical stimulation induces metamorphosis in the ascidian Ciona intestinalis type A

Wakai M.K., Nakamura M.J., Sawai S., Hotta K., Oka K.

Proceedings of the Royal Society B: Biological Sciences （Proceedings of the Royal Society B: Biological Sciences） 288 （ 1945 ） 2021年02月

ISSN 09628452

Marine invertebrate larvae are known to begin metamorphosis in response to environmentally derived cues. However, little is known about the relationships between the perception of such cues and internal signalling for metamorphosis. To elucidate the mechanism underlying the initiation of metamorphosis in the ascidian, Ciona intestinalis type A (Ciona robusta), we artificially induced ascidian metamorphosis and investigated Ca 2+ dynamics from pre- to post-metamorphosis. Ca 2+ transients were observed and consisted of two temporally distinct phases with different durations before tail regression which is the early event of metamorphosis. In the first phase, Phase I, the Ca 2+ transient in the papillae (adhesive organ of the anterior trunk) was coupled with the Ca 2+ transient in dorsally localized cells and endoderm cells just after mechanical stimulation. The Ca 2+ transients in Phase I were also observed when applying only short stimulation. In the second phase, Phase II, the Ca 2+ transient in papillae was observed again and lasted for approximately 5-11 min just after the Ca 2+ transient in Phase I continued for a few minutes. The impaired papillae by Foxg-knockdown failed to induce the second Ca 2+ transient in Phase II and tail regression. In Phase II, a wave-like Ca 2+ propagation was also observed across the entire epidermis. Our results indicate that the papillae sense a mechanical cue and two-round Ca 2+ transients in papillae transmits the internal metamorphic signals to different tissues, which subsequently induces tail regression. Our study will help elucidate the internal mechanism of metamorphosis in marine invertebrate larvae in response to environmental cues.

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Sexual and asexual development: two distinct programs producing the same tunicate

Kowarsky M., Anselmi C., Hotta K., Burighel P., Zaniolo G., Caicci F., Rosental B., Neff N.F., Ishizuka K.J., Palmeri K.J., Okamoto J., Gordon T., Weissman I.L., Quake S.R., Manni L., Voskoboynik A.

Cell Reports （Cell Reports） 34 （ 4 ） 2021年01月

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways.

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The ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis

Hotta K., Dauga D., Manni L.

Scientific Reports （Scientific Reports） 10 （ 1 ） 2020年12月

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

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Phylogenetic comparison of egg transparency in ascidians by hyperspectral imaging

Shito T.T., Hasegawa N., Oka K., Hotta K.

Scientific Reports （Scientific Reports） 10 （ 1 ） 2020年12月

The transparency of animals is an important biological feature. Ascidian eggs have various degrees of transparency, but this characteristic has not yet been measured quantitatively and comprehensively. In this study, we established a method for evaluating the transparency of eggs to first characterize the transparency of ascidian eggs across different species and to infer a phylogenetic relationship among multiple taxa in the class Ascidiacea. We measured the transmittance of 199 eggs from 21 individuals using a hyperspectral camera. The spectrum of the visual range of wavelengths (400–760 nm) varied among individuals and we calculated each average transmittance of the visual range as bio-transparency. When combined with phylogenetic analysis based on the nuclear 18S rRNA and the mitochondrial cytochrome c oxidase subunit I gene sequences, the bio-transparencies of 13 species were derived from four different families: Ascidiidae, Cionidae, Pyuridae, and Styelidae. The bio-transparency varied 10–90% and likely evolved independently in each family. Ascidiella aspersa showed extremely high (88.0 ± 1.6%) bio-transparency in eggs that was maintained in the “invisible” larva. In addition, it was indicated that species of the Ascidiidae family may have a phylogenetic constraint of egg transparency.

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Cellular thermogenesis compensates environmental temperature fluctuations for maintaining intracellular temperature

Yamanaka R., Shindo Y., Hotta K., Hiroi N., Oka K.

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 533 （ 1 ） 70 - 76 2020年11月

ISSN 0006291X

Temperature governs states and dynamics of all biological molecules, and several cellular processes are often heat sources and/or sinks. Technical achievement of intracellular thermometry enables us to measure intracellular temperature, and it can offer novel perspectives in biology and medicine. However, little is known that changes of intracellular temperature throughout the cell-cycle and the manner of which cells regulates their thermogenesis in response to fluctuation of the environmental temperature. Here, cell-cycle-dependent changes of intracellular temperature were reconstructed from the snapshots of cell population at single-cell resolution using ergodic analysis for asynchronously cultured HeLa cells expressing a genetically encoded thermometry. Intracellular temperature is highest at G1 phase, and it gradually decreases along cell-cycle progression and increases abruptly during mitosis. Cells easily heated up are harder to cool down and vice versa, especially at G1/S phases. Together, intracellular thermogenesis depends on cell-cycle phases and it maintains intracellular temperature through compensating environmental temperature fluctuations.

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Different strategies for tissue scaling in dwarf tailbud embryos revealed by single-cell analysis

Matsumura K.D., Nakamura M.J., Koizumi W.C., Hotta K., Oka K.

Developmental Biology （Developmental Biology） 460 （ 2 ） 215 - 223 2020年04月

ISSN 00121606

The tailbud stage is part of the organogenesis period—an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 μm (correspond to > 40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

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Neural properties of fundamental function encoding of sound selectivity in the female avian auditory cortex

Inda M., Hotta K., Oka K.

European Journal of Neuroscience （European Journal of Neuroscience） 51 （ 8 ） 1770 - 1783 2020年04月

ISSN 0953816X

Zebra finches (Taeniopygia guttata) use their voices for communication. Song structures in the songs of individual males are important for sound recognition in females. The caudomedial mesopallium (CMM) and nidopallium (NCM) are known to be essential higher auditory regions for sound recognition. These two regions have also been discussed with respect to their fundamental functions and song selectivity. To clarify their functions and selectivity, we investigated latencies and spiking patterns and also developed a novel correlation analysis to evaluate the relationship between neural activity and the characteristics of acoustic factors. We found that the latencies and spiking patterns in response to song stimuli differed between the CMM and NCM. In addition, our correlation analysis revealed that amplitude and frequency structures were important temporal acoustic factors for both regions. Although the CMM and NCM have different fundamental functions, they share similar encoding systems for acoustic factors.

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Optical Dissection of Synaptic Plasticity for Early Adaptation in Caenorhabditis elegans

Ashida K., Shidara H., Hotta K., Oka K.

Neuroscience （Neuroscience） 428 112 - 121 2020年01月

ISSN 03064522

To understand neuronal information processing, it is essential to investigate the input–output relationship and its modulation via detailed dissections of synaptic transmission between pre- and postsynaptic neurons. In Caenorhabditis elegans, pre-exposure to an odorant for five minutes reduces chemotaxis (early adaptation). AWC sensory neurons and AIY interneurons are crucial for this adaptation; AWC neurons sense volatile odors, and AIY interneurons receive glutamatergic inputs from AWC neurons. However, modulations via early adaptation of the input–output relationship between AWC and AIY are not well characterized. Here we use a variety of fluorescent imaging techniques to show that reduced synaptic-vesicle release without Ca2+ modulation in AWC neurons suppresses the Ca2+ response in AIY neurons via early adaptation. First, early adaptation modulates the Ca2+ response in AIY but not AWC neurons. Adaptation in the Ca2+ signal measured in AIY neurons is caused by adaptation in glutamate release from AWC neurons. Further, we found that a G protein γ-subunit, GPC-1, is related to modulation of glutamate input to AIY. Our results dissect the modulation of the pre- and postsynaptic relationship in vivo based on optical methods, and demonstrate the importance of neurotransmitter-release modulation in presynaptic neurons without Ca2+ modulation.

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Inhibition of mg2+ extrusion attenuates glutamate excitotoxicity in cultured rat hippocampal neurons

Shindo Y., Yamanaka R., Hotta K., Oka K.

Nutrients （Nutrients） 12 （ 9 ） 1 - 14 2020年

Magnesium plays important roles in the nervous system. An increase in the Mg2+ concentration in cerebrospinal fluid enhances neural functions, while Mg2+ deficiency is implicated in neuronal diseases in the central nervous system. We have previously demonstrated that high concentrations of glutamate induce excitotoxicity and elicit a transient increase in the intracellular concentration of Mg2+ due to the release of Mg2+ from mitochondria, followed by a decrease to below steady-state levels. Since Mg2+ deficiency is involved in neuronal diseases, this decrease presumably affects neuronal survival under excitotoxic conditions. However, the mechanism of the Mg2+ decrease and its effect on the excitotoxicity process have not been elucidated. In this study, we demonstrated that inhibitors of Mg2+ extrusion, quinidine and amiloride, attenuated glutamate excitotoxicity in cultured rat hippocampal neurons. A toxic concentration of glutamate induced both Mg2+ release from mitochondria and Mg2+ extrusion from cytosol, and both quinidine and amiloride suppressed only the extrusion. This resulted in the maintenance of a higher Mg2+ concentration in the cytosol than under steady-state conditions during the ten-minute exposure to glutamate. These inhibitors also attenuated the glutamate-induced depression of cellular energy metabolism. Our data indicate the importance of Mg2+ regulation in neuronal survival under excitotoxicity.

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Serotonin modulates behavior-related neural activity of RID interneuron in Caenorhabditis elegans

Mori H., Ashida K., Shidara H., Nikai T., Hotta K., Oka K.

PLoS ONE （PLoS ONE） 14 （ 12 ） 2019年12月

Animals change their behaviors in response to external stimuli, and numerous neurotransmitters are involved in these behavioral changes. In Caenorhabditis elegans, serotonin (5- HT) affects various behaviors such as inhibition of locomotion, stimulation of egg laying, and pharyngeal pumping. Previous research has shown that the neural activity of the RID interneuron increases when the worm moves forward, and the RID is necessary for sustaining forward locomotion. However, the relationship between 5-HT and neural activity of RID, and how it modulates the behavior of the worm has not been investigated. In this article, we reveal the relationship among 5-HT, RID activity, and the behavior of worms using a custom- made tracking and imaging system. We simultaneously measured the neural activity of the RID and behavior in worms with three conditions: mock animals, animals pre-exposed to 5-HT, and 5-HT receptor mod-1 mutants. As shown in previous research, the neural activity of the RID increased during the transition from backward to forward, whereas it decreased during the transition from forward to backward in mock animals. These changes in neural activity were not observed in animals pre-exposed to 5-HT and mod-1 mutants. Moreover, RID activity was correlated with the velocity of the worm in mock animals. However, this correlation was not observed in animals pre-exposed to 5-HT and mod-1 mutants. Our results demonstrate that 5-HT modulates the activity of the RID interneuron, and we infer that the RID plays a role in modulating forward locomotion by changing its activity through 5-HT.

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The Input-Output Relationship of AIY Interneurons in Caenorhabditis elegans in Noisy Environment

Ashida K., Hotta K., Oka K.

iScience （iScience） 19 191 - 203 2019年09月

Determining how neurotransmitter input causes various neuronal activities is crucial to understanding neuronal information processing. In Caenorhabditis elegans, AIY interneurons receive several sources of sensory information as glutamate inputs and regulate behavior by integrating these inputs. However, the relationship between glutamate input and the Ca2+ response in AIY under environmental noise, in other words, without explicit stimulation, remains unknown. Here, we show that glutamate-input fluctuations evoke a sporadic Ca2+ response in AIY without stimulation. To ensure that Ca2+ response can be considered AIY output, we show that the membrane-potential depolarization precedes Ca2+ responses in AIY. We used an odor as model stimulation to modulate the sensory inputs. Simultaneous imaging of glutamate input and Ca2+ response, together with glutamate transmission mutants, showed that glutamate-input fluctuations evoke sporadic Ca2+ responses. We identified the input-output relationships under environmental noise in vivo, and our results address the relationship between sensory-input fluctuations and behavioral variability. Neuroscience; Systems Neuroscience; Sensory Neuroscience

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Multiple tracking and machine learning reveal dopamine modulation for area-restricted foraging behaviors via velocity change in Caenorhabditis elegans

Ashida K., Kato T., Hotta K., Oka K.

Neuroscience Letters （Neuroscience Letters） 706 68 - 74 2019年07月

ISSN 03043940

Food exploration is an essential survival behavior in organisms. To find food efficiently, many organisms use a foraging strategy called area-restricted search (ARS) wherein individuals first turn more frequently, restricting their search to one area, then turn less frequently, moving along a straight path to widen the search area. Previous research suggests that the nematode Caenorhabditis elegans shows ARS behavior by changing turn frequency, and that dopamine is a crucial determinant. However, the effects of dopamine on multiple behavioral parameters have remained unknown. Here, we evaluated turn (pirouette) frequency, moving velocity, and specific area occupancy (cell occupancy) over time by using a multiple-worms tracking system. In the control (mock) experiments, all parameters changed over time, but no changes were observed in experiments with dopamine pre-exposed and dopamine-deficient animals. In inverse reinforcement learning analysis, the value function for specific velocity was found to modulate over time in mock animals only. These results demonstrate that dopamine regulates ARS via changes not only to pirouette frequency change but also to velocity.

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GABA-Induced Intracellular Mg 2+ Mobilization Integrates and Coordinates Cellular Information Processing for the Maturation of Neural Networks

Yamanaka R., Shindo Y., Hotta K., Suzuki K., Oka K.

Current Biology （Current Biology） 28 （ 24 ） 3984 - 3991.e5 2018年12月

ISSN 09609822

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14-3-3ϵa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis

Mizotani Y., Suzuki M., Hotta K., Watanabe H., Shiba K., Inaba K., Tashiro E., Oka K., Imoto M.

Proceedings of the National Academy of Sciences of the United States of America （Proceedings of the National Academy of Sciences of the United States of America） 115 （ 38 ） E8873 - E8881 2018年09月

ISSN 00278424

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3ϵa protein as a direct binding partner of UTKO1 and showed that 14-3-3ϵa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3ϵa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3ϵa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3ϵa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3ϵa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3ϵa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3ϵa or ERM, or in larvae expressing a truncated form of 14-3-3ϵa that lacks the ability to interact with ERM. These results suggest that 14-3-3ϵa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

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Developmental Roles and Evolutionary Significance of AMPA-Type Glutamate Receptors

Hirai S., Hotta K., Okado H.

BioEssays （BioEssays） 40 （ 9 ） 2018年09月

ISSN 02659247

Organogenesis and metamorphosis require the intricate orchestration of multiple types of cellular interactions and signaling pathways. Glutamate (Glu) is an excitatory extracellular signaling molecule in the nervous system, while Ca 2+ is a major intracellular signaling molecule. The first Glu receptors to be cloned are Ca 2+ -permeable receptors in mammalian brains. Although recent studies have focused on Glu signaling in synaptic mechanisms of the mammalian central nervous system, it is unclear how this signaling functions in development. Our recent article demonstrated that Ca 2+ -permeable AMPA-type Glu receptors (GluAs) are essential for formation of a photosensitive organ, development of some neurons, and metamorphosis, including tail absorption and body axis rotation, in ascidian embryos. Based on findings in these embryos and mammalian brains, we formed several hypotheses regarding the evolution of GluAs, the non-synaptic function of Glu, the origin of GluA-positive neurons, and the neuronal network that controls metamorphosis in ascidians.

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Effect of interactions among individuals on the chemotaxis behaviours of Caenorhabditis elegans

Yoshimizu, Toshiki Shidara, Hisashi Ashida, Keita Hotta, Kohji Oka, Kotaro

The Journal of Experimental Biology （The Journal of experimental biology） 221 （ 11 ） jeb182790 2018年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-0949

In many species, individual social animals interact with others in their group and change their collective behaviours. For the solitary nematode Caenorhabditis elegans strain N2, previous research suggests that individuals can change the behaviour of other worms via pheromones and mechanosensory interactions. In particular, pheromones affect foraging behaviour, so that the chemotactic behaviours of individuals in a group (population) can be modulated by interactions with other individuals in the population. To investigate this, we directly compared the chemotactic behaviours of isolated (single) worms with those of individual animals within a population. We found that worms approached an odour source in a distinct manner depending on whether they were alone or in a population. Analysis of behaviours of the N2 worm and a pheromone production-defective mutant revealed that the 'pirouette' strategy was modulated by interaction of the worms via pheromones. Thus, pheromones play an important role in the characteristic collective behaviours seen in the population condition.

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Characterization of calcium transients during early embryogenesis in ascidians Ciona robusta (Ciona intestinalis type A) and Ciona savignyi

Akahoshi, Taichi Hotta, Kohji Oka, Kotaro

Developmental Biology 431 （ 2 ） 205 - 214 2017年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1095564X

高感度カルシウムイオンインジケーターGCaMP6を用いてホヤ発生過程における一過的カルシウムイオン濃度上昇を長時間・高い時間解像度で網羅的に明らかにした。その結果、これまで中脳・後脳境界に存在すると考えられていた運動を出力するためのリズム生成子がより後方の運動神経節に存在すること、細胞外カルシウムイオン依存的に尾部表皮細胞のカルシウムイオン濃度上昇がみられることなど多くの新たな発生におけるカルシウムイオン濃度上昇の振る舞いを発見した。

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AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.

堀田耕司

Proc Natl Acad Sci U S A 114 （ 15 ） 3939 - 3944 2017年04月

研究論文（学術雑誌）, 共著, 査読有り

脊椎動物ではAMPA受容体が４つあり、すべてを取り除く実験が困難であったた。一方、ホヤはAMPA受容体を１つしかもたないため、ノックダウンによる表現型の解析が比較的容易である。今回、ホヤAMAPA受容体のノックダウンおよび変異AMAPA受容体遺伝子導入により、はじめてAMPA受容体の発生における新たな機能を発見した。ホヤのAMPA受容体は中枢神経系の一部にのみ発現しており、その発現を阻害したところ、哺乳類の松果体にあたる光受容感覚器の形成が阻害されたことに加え、成体への変態に必要な尾の退縮が不完全となり、体軸の回転が阻害さることがわかった。

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Compartmentalized cGMP responses of olfactory sensory neurons in Caenorhabditis elegans.

堀田耕司

Journal of Neuroscience 37 （ 14 ） 3753 - 3763 2017年04月

査読有り

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Transitional correlation between inner-membrane potential and ATP levels of neuronal mitochondria

Suzuki, R. Hotta, K. Oka, K.

Scientific Reports （Scientific Reports） 8 （ 1 ） 2017年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

The importance of highly active mitochondria and their contribution to neuronal function has been of recent interest. In most cases, however, mitochondrial activity is estimated using measurements of mitochondrial inner membrane potential (IMPmito), and little is known about the dynamics of native mitochondrial ATP (ATPmito). This study conducted simultaneous imaging of IMPmito and ATPmito in neurons to explore their behaviour and their correlation during physiological mitochondrial/neuronal activity. We found that mitochondrial size, transport velocity and transport direction are not dependent on ATPmito or IMPmito. However, changes in ATPmito and IMPmito during mitochondrial fission/fusion were found; IMPmito depolarized via mitochondrial fission and hyperpolarized via fusion, and ATPmito levels increased after fusion. Because the density of mitochondria is higher in growth cones (GCs) than in axonal processes, integrated ATPmito signals (density × ATPmito) were higher in GCs. This integrated signal in GCs correlated with axonal elongation. However, while the averaged IMPmito was relatively hyperpolarized in GCs, there was no correlation between IMPmito in GCs and axonal elongation. A detailed time-course analysis performed to clarify the reason for these discrepancies showed that IMPmito and ATPmito levels did not always correlate accurately; rather, there were several correlation patterns that changed over time.

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Altered expression of Mg²⁺ transport proteins during Parkinson's disease-like dopaminergic cell degeneration in PC12 cells

Shindo Yutaka, Yamanaka Ryu, Suzuki Koji, Hotta Kohji, Oka Kotaro

Biochimica et Biophysica Acta - Molecular Cell Research 1863 （ 8 ） 1979 - 1984 2016年08月

ISSN 0167-4889

Mg²⁺ is an essential cation to maintain cellular functions, and intracellular Mg²⁺ concentration ([Mg²⁺]_i) is regulated by Mg²⁺ channels and transporters. In our previous study, we demonstrated that MPP⁺ elicits Mg²⁺ influx across the cell membrane and Mg²⁺ mobilization from mitochondria, and the resulting [Mg²⁺]_i is an important determinants of the cell viability in MPP⁺ model of Parkinson's disease (PD). It indicates that cellular Mg²⁺ transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg²⁺ transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg²⁺ transport proteins upon the exposure to MPP⁺. In thirteen Mg²⁺ transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP⁺-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H₂O₂ and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP⁺, suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg²⁺ transport proteins is implicated in the MPP⁺ model of PD, and it affects cell degeneration.

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Mitochondrial Mg²⁺ homeostasis decides cellular energy metabolism and vulnerability to stress

Yamanaka Ryu, Tabata Sho, Shindo Yutaka, Hotta Kohji, Suzuki Koji, Soga Tomoyoshi, Oka Kotaro

Scientific Reports 6 2016年07月

ISSN 2045-2322

Cellular energy production processes are composed of many Mg²⁺ dependent enzymatic reactions. In fact, dysregulation of Mg²⁺ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg²⁺ stores. Several biological stimuli alter mitochondrial Mg²⁺ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg²⁺ alteration affect cellular energy metabolism remains unclear. Mg²⁺ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg²⁺ uptake system. Here, we comprehensively analyzed intracellular Mg²⁺ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg²⁺ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg²⁺ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg²⁺ level in response to physiological stimuli.

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An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

Shindo Yutaka, Komatsu Hirokazu, Hotta Kohji, Ariga Katsuhiko, Oka Kotaro

Scientific Reports 6 2016年07月

ISSN 2045-2322

Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu₃), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu₃ induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu₃-treated HeLa cells. This indicates that PBu₃ can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu₃-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu₃ mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu₃, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu₃. Moreover, the application and removal of PBu₃ reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu₃ enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells.

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Qualitative and quantitative estimation of comprehensive synaptic connectivity in short- and long-term cultured rat hippocampal neurons with new analytical methods inspired by Scatchard and Hill plots

Tanamoto Ryo, Shindo Yutaka, Niwano Mariko, Matsumoto Yoshinori, Miki Norihisa, Hotta Kohji, Oka Kotaro

Biochemical and Biophysical Research Communications 471 （ 4 ） 486 - 491 2016年03月

ISSN 0006-291X

To investigate comprehensive synaptic connectivity, we examined Ca²⁺ responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca²⁺ responses was low during the application of stepwise increase of electric current in short-term cultured neurons (less than 17 days in-vitro (DIV)). The neurons cultured over 17 DIV showed two-type responses: S-shaped (sigmoid) and monotonous saturated responses, and Scatchard plots well illustrated the difference of these two responses. Furthermore, sigmoid like neural network responses over 17 DIV were altered to the monotonous saturated ones by the application of the mixture of AP5 and CNQX, specific blockers of NMDA and AMPA receptors, respectively. This alternation was also characterized by the change of Hill coefficients. These findings indicate that the neural network with sigmoid-like responses has strong synergetic or cooperative synaptic connectivity via excitatory glutamate synapses.

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Neural depolarization triggers Mg²⁺ influx in rat hippocampal neurons

Yamanaka R., Shindo Y., Karube T., Hotta K., Suzuki K., Oka K.

Neuroscience 310 731 - 741 2015年12月

ISSN 0306-4522

Homeostasis of magnesium ion (Mg²⁺) plays key roles in healthy neuronal functions, and deficiency of Mg²⁺ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg²⁺ concentration ([Mg²⁺]_i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg²⁺]_i. The aim of this study is to explore the direct relationship between neural activity and [Mg²⁺]_i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg²⁺]_i and [Ca²⁺]_i dynamics were simultaneously visualized with a highly selective fluorescent Mg²⁺ probe, KMG-104, and a fluorescent Ca²⁺ probe, Fura Red, respectively. [Mg²⁺]_i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg²⁺]_i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K⁺ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca²⁺ increase. Furthermore, the [Mg²⁺]_i increase was abolished in Mg²⁺-free extracellular medium, indicating [Mg²⁺]_i increase is due to Mg²⁺ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na⁺ channel opener, induced [Mg²⁺]_i increase, and this [Mg²⁺]_i increase was suppressed by the pretreatment of a non-specific Mg²⁺ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg²⁺]_i increase results from Mg²⁺ influx through 2-APB-sensitive channels in rat hippocampal neurons.

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Intracellular magnesium level determines cell viability in the MPP⁺ model of Parkinson's disease

Shindo Yutaka, Yamanaka Ryu, Suzuki Koji, Hotta Kohji, Oka Kotaro

Biochimica et Biophysica Acta - Molecular Cell Research 1853 （ 12 ） 3182 - 3191 2015年12月

ISSN 0167-4889

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg²⁺ homeostasis. Mg²⁺ is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg²⁺ homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg²⁺ concentration ([Mg²⁺]_i) and the role of Mg²⁺ in PD have still been obscure. In this study, we investigated [Mg²⁺]_i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP⁺) model of PD in differentiated PC12 cells. Application of MPP⁺ induced an increase in [Mg²⁺]_i immediately via two different pathways: Mg²⁺ release from mitochondria and Mg²⁺ influx across cell membrane, and the increased [Mg²⁺]_i sustained for more than 16h after MPP⁺ application. Suppression of Mg²⁺ influx decreased the viability of the cells exposed to MPP⁺. The cell viability correlated highly with [Mg²⁺]_i. In the PC12 cells with suppressed Mg²⁺ influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP⁺. Our results indicate that the increase in [Mg²⁺]_i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP⁺ toxicity.

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Spatiotemporal quantification of subcellular ATP levels in a single HeLa cell during changes in morphology

Suzuki Rika, Hotta Kohji, Oka Kotaro

Scientific Reports 5 2015年11月

The quantitative relationship between change in cell shape and ATP consumption is an unsolved problem in cell biology. In this study, a simultaneous imaging and image processing analysis allowed us to observe and quantify these relationships under physiological conditions, for the first time. We focused on two marginal regions of cells: the microtubule-rich € lamella € and the actin-rich € peripheral structure €. Simultaneous imaging and correlation analysis revealed that microtubule dynamics cause lamellar shape change accompanying an increase in ATP level. Also, image processing and spatiotemporal quantification enabled to visualize a chronological change of the relationships between the protrusion length and ATP levels, and it suggested they are influencing each other. Furthermore, inhibition of microtubule dynamics diminished motility in the peripheral structure and the range of fluctuation of ATP level in the lamella. This work clearly demonstrates that cellular motility and morphology are regulated by ATP-related cooperative function between microtubule and actin dynamics.

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Electrical stimulation of cultured neurons using a simply patterned indium-tin-oxide (ITO) glass electrode

Tanamoto Ryo, Shindo Yutaka, Miki Norihisa, Matsumoto Yoshinori, Hotta Kohji, Oka Kotaro

Journal of Neuroscience Methods 253 272 - 278 2015年09月

ISSN 0165-0270

Background: Indium-tin-oxide (ITO) glass electrodes possess the properties of optical transparency and high electrical conductivity, which enables the electrical stimulation of cultured cells to be performed whilst also measuring the responses with fluorescent imaging techniques. However, the quantitative relationship between the intensity of the stimulating current and the cell response is unclear when using conventional methods that employ a separated configuration of counter and stimulation electrodes. New method: A quantitative electrical current stimulation device without the use of a counter electrode was fabricated. Results: Nerve growth factor (NGF)-induced differentiated PC12 cells were cultured on an ITO single glass electrode, and the Ca2+ response to electrical stimuli was measured using fluorescent Ca2+ imaging. ITO electrode devices with a width less than 0.1mm were found to evoke a Ca2+ response in the PC12 cells. Subsequent variation in the length of the device in the range of 2-10mm was found to have little influence on the efficiency of the electric stimulus. We found that the stimulation of the cells was dependent on the electrical current, when greater than 60μA, rather than on the Joule heat, regardless of the width and length of the conductive area. Comparison with existing method(s): Because of the cells directly in contact with the electrode, our device enables to stimulate the cells specifically, comparing with previous devices with the counter electrode. Conclusions: The ITO device without the use of a counter electrode is a useful tool for evaluating the quantitative neural excitability of cultured neurons.

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Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis

Mizotani Yuji, Itoh Shun, Hotta Kohji, Tashiro Etsu, Oka Kotaro, Imoto Masaya

Biochemical and Biophysical Research Communications 463 （ 4 ） 656 - 660 2015年05月

ISSN 0006-291X

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

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Comprehensive morphological analysis of individual peripheral neuron dendritic arbors in ascidian larvae using the photoconvertible protein kaede

Yokoyama Takatoshi D., Hotta Kohji, Oka Kotaro

Developmental Dynamics 243 （ 10 ） 1362 - 1373 2014年10月

ISSN 1058-8388

Background: Ascidian larval epidermal sensory neurons (ESNs) each extend a single cilium into the outer body structure, or tunic, to form a unique external sensory network, the ASNET (ascidian dendritic network in tunic). The functions of ESNs and the ASNET are unknown, though it has been suggested that they play important roles in swimming larvae. In this study, we used transgenic larva expressing the photoconvertible protein Kaede pan-neuronally to identify the orientation and morphology of single ESN external processes within the ASNET. Results: When individual ESN cell bodies were UV-irradiated, the photoconverted Kaede protein diffused into the external processes that projected to the edge of the tunic, indicating that these processes are cytoplasmically connected to the cell bodies of ESNs. We were, therefore, able to comprehensively catalog the morphology and orientation of each ESN external process. Most trunk ESNs appeared to extend neurites to the palp, but no processes were observed to emanate from palp neurons. ESN processes differed systematically in their morphology and orientation, suggesting that the ASNET is formed in a regulated, non-random fashion. Conclusions: This study reveals the organization of ESN sensory fields in the ascidian larval tunic.

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Identification of oscillatory firing neurons associated with locomotion in the earthworm through synapse imaging

Shimoi T., Mizutani K., Kojima D., Kitamura Y., Hotta K., Ogawa H., Oka K.

Neuroscience 268 149 - 158 2014年05月

ISSN 0306-4522

We used FM imaging to identify neurons that receive sensory feedback from the body wall in a circuit for octopamine (OA)-evoked rhythmic locomotion in the earthworm, Eisenia fetida. We visualized synapses in which postsynaptic neurons receive the sensory feedback, by using FM1-43 dye to label the synapses of both motor and sensory pathways that are associated with locomotion, then clearing the motor pathway synapse labeling, and finally identifying the target synapses by distinguishing physiologically functional synapses through destaining using a high-K⁺ solution. A pair of synaptic regions associated with the sensory feedback was found to be located two or three cell body-widths away from the midline, between the anterior parts of the roots of the second lateral nerves (LNs) at the segmental ganglia (SGs). Using conventional intracellular recording and dye loading of the cell bodies surrounding these synaptic regions, we identified a pair of bilateral neurons with cell bodies larger than those of other cells in these regions, and named them "Oscillatory firing neurons Projecting to Peripheral nerves" (OPPs). These had a bipolar shape and projected neurites to the ipsilateral first and third LNs, fired rhythmically, and had a burst timing synchronized with the motor pattern bursts from the ipsilateral first LNs. Current injection into an OPP caused firing in the ipsilateral first LNs, supporting the hypothesis that OPPs functionally project to the peripheral nerves. OPPs also sent neurites to the adjacent anterior and posterior SGs, suggesting connections with the adjacent segments. We conclude that FM imaging can be used to identify neurons involved in specific functions, and that OPPs are the first neurons to be associated with OA-induced locomotion in the earthworm. © 2014 .

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Ontology for the asexual development and anatomy of the colonial chordate botryllus schlosseri

Manni Lucia, Gasparini Fabio, Hotta Kohji, Ishizuka Katherine J., Ricci Lorenzo, Tiozzo Stefano, Voskoboynik Ayelet, Dauga Delphine

PLoS One 9 （ 5 ） 2014年05月

Ontologies provide an important resource to integrate information. For developmental biology and comparative anatomy studies, ontologies of a species are used to formalize and annotate data that are related to anatomical structures, their lineage and timing of development. Here, we have constructed the first ontology for anatomy and asexual development (blastogenesis) of a bilaterian, the colonial tunicate Botryllus schlosseri. Tunicates, like Botryllus schlosseri, are non-vertebrates and the only chordate taxon species that reproduce both sexually and asexually. Their tadpole larval stage possesses structures characteristic of all chordates, i.e. a notochord, a dorsal neural tube, and gill slits. Larvae settle and metamorphose into individuals that are either solitary or colonial. The latter reproduce both sexually and asexually and these two reproductive modes lead to essentially the same adult body plan. The Botryllus schlosseri Ontology of Development and Anatomy (BODA) will facilitate the comparison between both types of development. BODA uses the rules defined by the Open Biomedical Ontologies Foundry. It is based on studies that investigate the anatomy, blastogenesis and regeneration of this organism. BODA features allow the users to easily search and identify anatomical structures in the colony, to define the developmental stage, and to follow the morphogenetic events of a tissue and/or organ of interest throughout asexual development. We invite the scientific community to use this resource as a reference for the anatomy and developmental ontology of B. schlosseri and encourage recommendations for updates and improvements. © 2014 Manni et al.

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Design and synthesis of a FlAsH-type Mg²⁺ fluorescent probe for specific protein labeling

Fujii Tomohiko, Shindo Yutaka, Hotta Kohji, Citterio Daniel, Nishiyama Shigeru, Suzuki Koji, Oka Kotaro

Journal of the American Chemical Society 136 （ 6 ） 2374 - 2381 2014年02月

ISSN 0002-7863

Although the magnesium ion (Mg²⁺) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg²⁺ probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg ²⁺ probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg²⁺ concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ_em/max = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg²⁺, and the probe is highly selective for Mg²⁺ (K_d/Mg = 1.7 mM, K_d/Ca 100 mM). Application of the probe for imaging of Mg²⁺ in HeLa cells showed that this FlAsH-type Mg²⁺ sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg²⁺ concentration caused by the release of Mg²⁺ from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg²⁺. © 2014 American Chemical Society.

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A method for selective ablation of neurons in C. elegans using the phototoxic fluorescent protein, KillerRed

Kobayashi Junya, Shidara Hisashi, Morisawa Yuma, Kawakami Maki, Tanahashi Yuta, Hotta Kohji, Oka Kotaro

Neuroscience Letters 548 261 - 264 2013年08月

ISSN 0304-3940

Specific neuron ablation with laser microbeam has been used in behavioral analysis of Caenorhabditis elegans. However, this method is hard to acquire many ablated worms, and is unable to compare behavioral changes just before and after ablation. Here, we developed an ablation method by using genetically encoded photosensitizer protein, KillerRed, which produces reactive oxygen species by green light irradiation. Ablation of AWA sensory neurons abolished the chemotaxis to AWA specific sensitive attractant, diacetyl, and no functional effect on the other sensory neuron, AWC, which senses benzaldehyde. This ablation method can be useful for analyzing neural in situ. © 2013 Elsevier Ireland Ltd.

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Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

Nakamura Mitsuru J., Hotta Kohji, Oka Kotaro

PLoS One 8 （ 8 ） 2013年08月

Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm^-1, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis. © 2013 Nakamura et al.

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NO/cGMP/PKG signaling pathway induces magnesium release mediated by mitoK_ATP channel opening in rat hippocampal neurons

Yamanaka Ryu, Shindo Yutaka, Hotta Kohji, Suzuki Koji, Oka Kotaro

FEBS Letters 587 （ 16 ） 2643 - 2648 2013年08月

ISSN 0014-5793

Intracellular Mg²⁺ concentration ([_Mg2+]i) and NO regulate cell survival and death. To reveal the involvement of NO in intracellular Mg²⁺ regulation, we visualized intracellular Mg ²⁺ using the fluorescent Mg²⁺ indicator KMG-104-AM in rat hippocampal neurons. Pharmacological experiments using SNAP, 8-Br-cGMP, diazoxide and several inhibitors revealed that the NO/cGMP/Protein kinsase G (PKG) signaling pathway triggers an increase in [_Mg2+]i, and that Mg²⁺ mobilization is due to Mg²⁺ release from mitochondria induced by mitoK_ATP channel opening. In addition, Mg²⁺ release is potentiated by the positive feedback loop including mitoK_ATP channel opening, mitochondrial depolarization and PKC activation. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans

Shidara Hisashi, Kobayashi Junya, Tanamoto Ryo, Hotta Kohji, Oka Kotaro

Neuroscience Letters 541 199 - 203 2013年04月

ISSN 0304-3940

Although some interneurons in C. elegans have been shown to have unusual region-specific Ca²⁺ dynamics, the region-specific Ca²⁺ and membrane potential response properties of these neurons are largely unknown due to technical limitations. In this report, we focused on one of these neurons, AIY interneuron, where Ca²⁺ dynamics have been detected only in neurites, and not the soma, during odor and temperature stimulation to determine whether membrane potential and Ca²⁺ are region-specific dynamics and distinct from one another. To visualize voltage change both in the soma and neurites of AIY, we used voltage-sensitive fluorescent protein (VSFP) 2.42. First, we confirmed that the sensor protein worked correctly in C. elegans by depolarizing AIY interneuron with high concentrations of KCl. Next, we observed membrane potential depolarization during odor (isoamyl alcohol) stimulation in both neurites and the soma. Additionally, depolarization of membrane potential with direct application of high KCl induced a Ca²⁺ increase in the soma. From these results, we conclude that membrane potential behavior and Ca²⁺ dynamics in AIY differ in its subcellular regions and that VSFP2.42 can be a useful tool for studying information processing in single neurons. © 2013 Elsevier Ireland Ltd.

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Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans.

堀田耕司

Neuroscience letters Epub ahead of print 2013年02月

研究論文（学術雑誌）, 共著, 査読有り

Crosstalk between second messengers predicts the motility of the growth cone

Kobayashi Takahiko, Nagase Fumiaki, Hotta Kohji, Oka Kotaro

Scientific Reports 3 2013年

Axon guidance involves multiple second messenger signal transduction pathways. Although each signal transduction pathway has been characterized, only a few studies have examined crosstalk between these cascades. Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca 2+. The levels of cAMP and cGMP in non-stimulated freely extending growth cones showed a negative correlation without delay. Although there was no direct correlation between cAMP and Ca 2+, examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa. Furthermore, spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly. These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

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Three-dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution

Nakamura Mitsuru J., Terai Jun, Okubo Reiko, Hotta Kohji, Oka Kotaro

Developmental Biology 372 （ 2 ） 274 - 284 2012年12月

ISSN 0012-1606

During embryogenesis, chordates pass through a tailbud stage in which the larval tail is formed. Since acquisition of a tadpole-like tail during tailbud stage is one of the key events in the evolution of chordates, understanding the anatomy of the tailbud stage chordate embryo is of special interest. In this study, to understand comprehensively the anatomy of the tailbud embryo at single-cell-level, real microscopic image stacks of the tailbud embryo in Ciona intestinalis were reconstructed into a 3D computer model. This comprehensive 3D model of the ascidian tailbud embryo was based on real images of confocal laser scanning microscope (CLSM) and therefore, cell shape, location and cell arrangement reflect real geometries of the tailbud embryo. We found that the tailbud embryo consists of 1579 cells, including 836 epidermal cells, 228 cells in the central nervous system, 218 mesenchymal cells, four trunk ventral cells, two B/B^*8.11 cells, 36 muscle cells, 40 notochord cells, four primordial germ cells, and 199 endodermal cells. Moreover, we identified for the first time two populations of previously undefined cells (a total of 12 cells) in Ciona: one located in the lateral trunk and the other located under the tail dorsal epidermis. This information provides a first step for understanding how the body plan of the chordate tailbud embryo formed and evolved. © 2012 Elsevier Inc.

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Three-dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution.

堀田耕司

Developmental Biology （ 372 ） 274-284 2012年12月

研究論文（学術雑誌）, 共著, 査読有り

Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis

Kawada Tsuyoshi, Ogasawara Michio, Sekiguchi Toshio, Aoyama Masato, Hotta Kohji, Oka Kotaro, Satake Honoo

Endocrinology 152 （ 6 ） 2416 - 2427 2011年06月

ISSN 0013-7227

The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomic analysis detected 33 peptides, including 26 novel peptides, from C. intestinalis. The ascidian peptides are largely classified into three categories: 1) prototypes and homologs of vertebrate peptides, such as galanin/galanin-like peptide, which have never been identified in any invertebrates; 2) peptides partially homologous with vertebrate peptides, including novel neurotesin-like peptides; 3) novel peptides. These results not only provide evidence that C. intestinalis possesses various homologs and prototypes of vertebrate neuropeptides and peptide hormones but also suggest that several of these peptides might have diverged in the ascidian-specific evolutionary lineage. All Ciona peptide genes were expressed in the neural complex, where as several peptide gene transcripts were also distributed in peripheral tissues, including the ovary. Furthermore, a Ciona neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was shown to down-regulate growth of Ciona vitellogenic oocytes. These results suggest that the Ciona peptides act not only as neuropeptides in the neural tissue but also as hormones in nonneuronal tissues and that ascidians, unlike other invertebrates, such as nematodes, insects, and sea urchins, established an evolutionary origin of the peptidergic neuroendocrine, endocrine, and nervous systems of vertebrates with certain specific molecular diversity. Copyright © 2011 by The Endocrine Society.

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Chromatophore Activity during Natural Pattern Expression by the Squid Sepioteuthis lessoniana: Contributions of Miniature Oscillation

Mamiko Suzuki, Tetsuya Kimura, Hiroto Ogawa, Kohji Hotta, Kotaro Oka

PLoS One epub 2011年04月

研究論文（学術雑誌）, 共著, 査読有り

CIPRO 2.5

Endo Toshinori, Ueno Keisuke, Yonezawa Kouki, Mineta Katsuhiko, Hotta Kohji, Satou Yutaka, Yamada Lixy, Ogasawara Michio, Takahashi Hiroki, Nakajima Ayako, Nakachi Mia, Nomura Mamoru, Yaguchi Junko, Sasakura Yasunori, Yamasaki Chisato, Sera Miho, Yoshizawa Akiyasu C., Imanishi Tadashi, Taniguchi Hisaaki, Inaba Kazuo

Nucleic Acids Research 39 （ SUPPL. 1 ） 2011年01月

ISSN 0305-1048

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includesoriginal large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite ofdevelopmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and geneexpression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analysesat various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a communitysupported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http:// cipro.ibio.jp/2.5. © The Author(s) 2010.

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CIPRO 2.5: Ciona intestinalis Protein Database, a unique integrated repository of large-scale omics data, bioinformatic analyses, and curated annotation, with user rating and reviewing functionality

Endo, Toshinori; Ueno, Keisuke; Yonezawa, Kouki; Mineta, Katsuhiko; Hotta, Kohji; Satou, Yutaka; Yamada, Lixy; Ogasawara, Michio; Takahashi, Hiroki.; Nakajima, Ayako; Nakachi, Mia; Nomura, Mamoru; Yaguchi, Junko; Sasakura , Yasunori; Yamasaki, Chisato; Sera, Miho; Yoshizawa, Akiyasu; Imanishi, Tadashi; Taniguchi, Hisaaki; Inaba, Kazuo

Nucleic Acid Research 39 （ 1 ） 807-814 2011年01月

研究論文（学術雑誌）, 共著, 査読有り

Newly developed Mg ²⁺-selective fluorescent probe enables visualization of Mg ²⁺ dynamics in mitochondria

Shindo Yutaka, Fujii Tomohiko, Komatsu Hirokazu, Citterio Daniel, Hotta Kohji, Suzuki Koji, Oka Kotaro

PLoS One 6 （ 8 ） 2011年

Mg ²⁺ plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg ²⁺ regulation and the Mg ²⁺ concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg ²⁺ in mitochondria in intact cells. Here, we have developed a novel Mg ²⁺-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg ²⁺ concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg ²⁺, KMG-104, enabled us to compare the dynamics of Mg ²⁺ in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg ²⁺ mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg ²⁺ concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg ²⁺ and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP ⁺). A gradual decrease in the Mg ²⁺ concentration in mitochondria was observed in response to MPP ⁺ in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg ²⁺ dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)). © 2011 Shindo et al.

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Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis: homologs and prototypes of vertebrate peptides and novel peptides

Tsuyoshi Kawada, Michio Ogasawara, Toshio Sekiguchi, Masato Aoyama, Kohji Hotta, Kotaro Oka, Honoo Satake

Endocrinology Epub （ ahead ） of print 2011年

研究論文（学術雑誌）, 共著, 査読有り

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria.

Shindo Yutaka, Fujii Tomohiko, Komatsu Hirokazu, Citterio Daniel, Hotta Kohji, Suzuki Koji, Oka Kotaro

PLoS One 6 （ 8 ） 2011年

Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

Chromatophore activity during natural pattern expression by the squid Sepioteuthis lessoniana

Suzuki Mamiko, Kimura Tetsuya, Ogawa Hiroto, Hotta Kohji, Oka Kotaro

PLoS One 6 （ 4 ） 2011年

Squid can rapidly change the chromatic patterns on their body. The patterns are created by the expansion and retraction of chromatophores. The chromatophore consists of a central pigment-containing cell surrounded by radial muscles that are controlled by motor neurons located in the central nervous system (CNS). In this study we used semi-intact squid (Sepioteuthis lessoniana) displaying centrally controlled natural patterns to analyze spatial and temporal activities of chromatophores located on the dorsal mantle skin. We found that chromatophores oscillated with miniature expansions/retractions at various frequencies, even when the chromatic patterns appear macroscopically stable. The frequencies of this miniature oscillation differed between "feature" and "background" areas of chromatic patterns. Higher frequencies occurred in feature areas, whereas lower frequencies were detected in background areas. We also observed synchronization of the oscillation during chromatic pattern expression. The expansion size of chromatophores oscillating at high frequency correlated with the number of synchronized chromatophores but not the oscillation frequency. Miniature oscillations were not observed in denervated chromatophores. These results suggest that miniature oscillations of chromatophores are driven by motor neuronal activities in the CNS and that frequency and synchrony of this oscillation determine the chromatic pattern and the expansion size, respectively. © 2011 Suzuki et al.

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ACBD: Database for Ascidian Chemical Genomics.

Hira, Yuichiro, Terai, Jun, Imoto, Masaya, Tashiro, Etsu, and Hotta, Kohji.

Nature Precedings - （ - ） - 2010年12月

研究論文（学術雑誌）, 共著

Glutamate-induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Shindo Yutaka, Fujimoto Ai, Hotta Kohji, Suzuki Koji, Oka Kotaro

Journal of Neuroscience Research 88 （ 14 ） 3125 - 3132 2010年11月

ISSN 0360-4012

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG-104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate-induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate-induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate-induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley-Liss, Inc.

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Glutamate-Induced Calcium Increase Mediates Magnesium Release from Mitochondria in Rat Hippocampal Neurons

Yutaka Shindo, Ai Fujimoto, Kohji Hotta, Koji Suzuki and Kotaro Oka

Journal of Neuroscience Research. 88 （ 14 ） 3125-32 2010年11月

研究論文（学術雑誌）, 共著, 査読有り

The ANISEED database

Tassy Olivier, Dauga Delphine, Daian Fabrice, Sobral Daniel, Robin François, Khoueiry Pierre, Salgado David, Fox Vanessa, Caillol Danièle, Schiappa Renaud, Laporte Baptiste, Rios Anne, Luxardi Guillaume, Kusakabe Takehiro, Joly Jean Stéphane, Darras Sébastien, Christiaen Lionel, Contensin Magali, Auger Hélène, Lamy Clément, Hudson Clare, Rothbächer Ute, Gilchrist Michael J., Makabe Kazuhiro W., Hotta Kohji, Fujiwara Shigeki, Satoh Nori, Satou Yutaka, Lemaire Patrick

Genome Research 20 （ 10 ） 1459 - 1468 2010年10月

ISSN 1088-9051

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions. © 2010 by Cold Spring Harbor Laboratory Press.

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The ANISEED database: digital representation, formalisation and elucidation of a chordate developmental program

O. Tassy, D. Dauga, F. Daian, D. Sobral, F. Robin, P. Khoueiry, D. Salgado, V. Fox, D. Caillo, M. Contensin, Renaud Schiappa, Anne Rios, Guillaume Luxardi, M. Gilchrist, K. Makabe, K. Hotta, Fujiwara, T. Kusakabe, N. Satoh, Y. Satou and P. Lemaire

Genome Research. 20 （ 10 ） 1459-68 2010年10月

研究論文（学術雑誌）, 単著, 査読有り

Network structure of projections extending from peripheral neurons in tunic of ascidian larva

Hiroshi Q.Terakubo, Yoko Nakajima, Yasunori Sasakura, Takeo Horie, Alu Konno, Hiroki Takahashi, Kazuo Inaba, Kohji Hotta and Kotaro Oka

Developmental Dynamics. 239 （ 8 ） 2278-87 2010年08月

研究論文（学術雑誌）, 単著, 査読有り

Network structure of projections extending from peripheral neurons in the tunic of ascidian larva

Terakubo Hiroshi Q., Nakajima Yoko, Sasakura Yasunori, Horie Takeo, Konno Alu, Takahashi Hiroki, Inaba Kazuo, Hotta Kohji, Oka Kotaro

Developmental Dynamics 239 （ 8 ） 2278 - 2287 2010年08月

ISSN 1058-8388

In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole-larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 9+2 axonemes were accompanied by a vesicle-containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle-containing fibrous mass and no 9+2 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic. © 2010 Wiley-Liss, Inc.

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Blue fluorescent cGMP sensor for multiparameter fluorescence imaging

Niino Yusuke, Hotta Kohji, Oka Kotaro

PLoS One 5 （ 2 ） 2010年02月

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca ²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca ²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks. © 2010 Niino et al.

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Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos

Takahashi Hiroki, Hotta Kohji, Takagi Chiyo, Ueno Naoto, Satoh Nori, Shoguchi Eiichi

Zoological Science 27 （ 2 ） 110 - 118 2010年02月

ISSN 0289-0003

Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified ∼450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within ∼3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region ∼0.51.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates. © 2010 Zoological Society of Japan.

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Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos

Hiroki Takahashi1, Kohji Hotta, Chiyo Takagi, Naoto Ueno, Nori Satoh, Eiichi Shoguchi

Zoological Sci., 27 （ 2 ） 110-118 2010年02月

研究論文（学術雑誌）, 共著, 査読有り

Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Yusuke Niino, Kohji Hotta and Kotaro Oka

PLoS One. 5 （ 2 ） e9164 2010年02月

研究論文（学術雑誌）, 単著, 査読有り

Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis

Konno Alu, Kaizu Maiko, Hotta Kohji, Horie Takeo, Sasakura Yasunori, Ikeo Kazuho, Inaba Kazuo

Developmental Biology 337 （ 1 ） 42 - 62 2010年01月

ISSN 0012-1606

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9 + 0 structure similar to vertebrate primary cilia, a 9 + 0 structure with electron-dense materials in the center, a 9 + 2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan. © 2009 Elsevier Inc. All rights reserved.

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Distribution and structural diversity of cilia in the tadpole larvae of the ascidian Ciona intestinalis

Alu Konno, Maiko Kaizu, Kohji Hotta, Takeo Horie, Yasunori Sasakura, Kazuho Ikeo and Kazuo Inaba

Developmental Biology 1 （ 337 ） 42-62 2010年01月

研究論文（学術雑誌）, 共著, 査読有り

FMRFamide elicits chromatophore expansion and retraction depending on its type and development in the squid, Sepioteuthis lessoniana

Suzuki Mamiko, Kimura Tetsuya, Ogawa Hiroto, Hotta Kohji, Oka Kotaro

Invertebrate Neuroscience 9 （ 3-4 ） 185 - 193 2009年12月

ISSN 1354-2516

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development. © 2010 Springer-Verlag.

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Simultaneous live cell imaging using dual FRET sensors with a single excitation light

Niino Yusuke, Hotta Kohji, Oka Kotaro

PLoS One 4 （ 6 ） 2009年06月

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca²⁺ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility. © 2009 Niino et al.

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Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

Yamada Shigehiro, Hotta Kohji, Yamamoto Takamasa S., Ueno Naoto, Satoh Nori, Takahashi Hiroki

Developmental Biology 328 （ 1 ） 1 - 12 2009年04月

ISSN 0012-1606

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS. © 2009 Elsevier Inc. All rights reserved.

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Genome-wide searching with base-pairing kernel functions for noncoding RNAs

Morita Kensuke, Saito Yutaka, Sato Kengo, Oka Kotaro, Hotta Kohji, Sakakibara Yasubumi

Nucleic Acids Research 37 （ 3 ） 999 - 1009 2009年

ISSN 0305-1048

Despite the accumulating research on noncoding RNAs (ncRNAs), it is likely that we are seeing only the tip of the iceberg regarding our understanding of the functions and the regulatory roles served by ncRNAs in cellular metabolism, pathogenesis and host-pathogen interactions. Therefore, more powerful computational and experimental tools for analyzing ncRNAs need to be developed. To this end, we propose novel kernel functions, called base-pairing profile local alignment (BPLA) kernels, for analyzing functional ncRNA sequences using support vector machines (SVMs). We extend the local alignment kernels for amino acid sequences in order to handle RNA sequences by using STRAL's; scoring function, which takes into account sequence similarities as well as upstream and downstream base-pairing probabilities, thus enabling us to model secondary structures of RNA sequences. As a test of the performance of BPLA kernels, we applied our kernels to the problem of discriminating members of an RNA family from nonmembers using SVMs. The results indicated that the discrimination ability of our kernels is stronger than that of other existing methods. Furthermore, we demonstrated the applicability of our kernels to the problem of genome-wide search of snoRNA families in the Caenorhabditis elegans genome, and confirmed that the expression is valid in 14 out of 48 of our predicted candidates by using qRT-PCR. Finally, highly expressed six candidates were identified as the original target regions by DNA sequencing. © 2009 The Author(s).

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Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis

Satou Yutaka, Mineta Katsuhiko, Ogasawara Michio, Sasakura Yasunori, Shoguchi Eiichi, Ueno Keisuke, Yamada Lixy, Matsumoto Jun, Wasserscheid Jessica, Dewar Ken, Wiley Graham B., Macmil Simone L., Roe Bruce A., Zeller Robert W., Hastings Kenneth E M, Lemaire Patrick, Lindquist Erika, Endo Toshinori, Hotta Kohji, Inaba Kazuo

Genome Biology 9 （ 10 ） 2008年10月

ISSN 1474-7596

Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly. Results: We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5′-ends were precisely mapped using 5′-full-length ESTs, an important refinement even in otherwise unchanged models. Conclusion: Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever. © 2008 Satou et al.; licensee BioMed Central Ltd.

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A novel biological role of tachykinins as an up-regulator of oocyte growth: Identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis

堀田耕司

ENDOCRINOLOGY 149 （ 9 ） 4346 - 4356 2008年09月

研究論文（学術雑誌）, 共著, ISSN 0013-7227

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Ca²⁺ influx through P2X receptors induces actin cytoskeleton reorganization by the formation of cofilin rods in neurites

Homma Kohei, Niino Yusuke, Hotta Kohji, Oka Kotaro

Molecular and Cellular Neuroscience 37 （ 2 ） 261 - 270 2008年02月

ISSN 1044-7431

In physiological and pathological events, extracellular ATP plays an important role by controlling several types of purinergic receptors and changing cytoskeleton dynamics. To know the process of ATP-dependent cytoskeleton remodeling, we focused on cofilin, a key regulator of actin cytoskeleton, and investigated the dynamics of cofilin in PC12 cells through fluorescent protein-labeled cofilin and actin, Ca²⁺ imaging, and fluorescence resonance energy transfer (FRET) techniques. As a result, ATP induced intracellular Ca²⁺ increase, following cofilin rods' formation. ATP-induced cofilin rods' formation was not observed in cells expressing unphosphorylatable variant of cofilin. A P2X receptor agonist, but not P2Y, induced the formation of cofilin rods, whereas calmodulin and calcineurin inhibitors suppressed it. These results indicate that Ca²⁺ influx through P2X receptors induces the formation of cofilin rods via calcineurin-dependent dephosphorylation of cofilin. This pathway might be one candidate to explain the effects of ATP on neuronal development and injury. © 2007 Elsevier Inc. All rights reserved.

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Brachyury-downstream gene sets in a chordate, Ciona intestinalis

Hotta Kohji, Takahashi Hiroki, Satoh Nori, Gojobori Takashi

Evolution and Development 10 （ 1 ） 37 - 51 2008年01月

ISSN 1520-541X

In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: Ciona Notochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, NfκB and TGF-β signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates. © 2008 The Author(s).

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A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva

堀田耕司

DEVELOPMENTAL DYNAMICS 236 （ 7 ） 1790 - 1805 2007年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1058-8388

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Brachyury-downstream notochord genes and convergent extension in Ciona intestinalis embryos

Hotta Kohji, Yamada Shigehiro, Ueno Naoto, Satoh Nori, Takahashi Hiroki

Development Growth and Differentiation 49 （ 5 ） 373 - 382 2007年06月

ISSN 0012-1592

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo. © 2007 The Authors.

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T-box genes in the Ascidian Ciona intestinalis

Takatori Naohito, Hotta Kohji, Mochizuki Yasuaki, Satoh Gouki, Mitani Yasuo, Satoh Nori, Satou Yutaka, Takahashi Hiroki

Developmental Dynamics 230 （ 4 ） 743 - 753 2004年08月

ISSN 1058-8388

Members of the T-box family of transcription factors share an evolutionary conserved DNA-binding domain and play significant roles in various processes of embryonic development. Vertebrate T-box genes are categorized into the following five major subfamilies (eight groups), depending on sequence similarities: Brachyury, Tbx1 (Tbx1/10, Tbx15/18/22, Tbx20), Tbx2/3/4/5 (Tbx2/3 and Tbx4/5), Tbx6, and Tbr/Eomes/TBX21. Ascidians are primitive chordates, and their tadpole larva are considered to represent the simplified and basic body plan of vertebrates. In addition, it has been revealed that the ascidian genome contains the basic ancestral complement of genes involved in development. The present characterization of cDNAs and survey of the Ciona intestinalis draft genome demonstrated that the Ciona genome contains a single copy gene for each of the Brachyury, Tbx1/10, Tbx15/18/22, Tbx20, Tbx2/3, and Tbr/Eomes/TBX21 groups, and at least three copies of the Tbx6 subfamily. Each of the Ciona T-box genes shows a characteristic expression pattern, although that of Tbx20 was not determined in the present study. These results provide basic information that will be useful for future studies of the function of each gene, genetic cascades of different T-box genes, and genome-wide surveys of evolutionary changes in the T-box gene structure and organization in this primitive chordate. © 2004 Wiley-Liss, Inc.

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A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Hotta Kohji, Takahashi Hiroki, Ueno Naoto, Gojobori Takashi

Gene 317 （ 1-2 ） 165 - 185 2003年10月

ISSN 0378-1119

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway. © 2003 Elsevier B.V. All rights reserved.

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The draft genome of Ciona intestinalis

Dehal Paramvir, Satou Yutaka, Campbell Robert K., Chapman Jarrod, Degnan Bernard, De Tomaso Anthony, Davidson Brad, Di Gregorio Anna, Gelpke Maarten, Goodstein David M., Harafuji Naoe, Hastings Kenneth E M, Ho Isaac, Hotta Kohji, Huang Wayne, Kawashima Takeshi, Lemaire Patrick, Martinez Diego, Meinertzhagen Ian A., Necula Simona, Nonaka Masaru, Putnam Nik, Rash Sam, Saiga Hidetoshi, Satake Masanobu, Terry Astrid, Yamada Lixy, Wang Hong Gang, Awazu Satoko, Azumi Kaoru, Boore Jeffrey, Branno Margherita, Chin-bow Stephen, DeSantis Rosaria, Doyle Sharon, Francino Pilar, Keys David N., Haga Shinobu, Hayashi Hiroko, Hino Kyosuke, Imai Kaoru S., Inaba Kazuo, Kano Shungo, Kobayashi Kenji, Kobayashi Mari, Lee Byung In, Makabe Kazuhiro W., Manohar Chitra, Matassi Giorgio, Medina Monica, Mochizuki Yasuaki, Mount Steve, Morishita Tomomi, Miura Sachiko, Nakayama Akie, Nishizaka Satoko, Nomoto Hisayo, Ohta Fumiko, Oishi Kazuko, Rigoutsos Isidore, Sano Masako, Sasaki Akane, Sasakura Yasunori, Shoguchi Eiichi, Shin-i Tadasu, Spagnuolo Antoinetta, Stainier Didier, Suzuki Miho M., Tassy Olivier, Takatori Naohito, Tokuoka Miki, Yagi Kasumi, Yoshizaki Fumiko, Wada Shuichi, Zhang Cindy, Hyatt P. Douglas, Larimer Frank, Detter Chris, Doggett Norman, Glavina Tijana, Hawkins Trevor, Richardson Paul, Lucas Susan, Kohara Yuji, Levine Michael, Satoh Nori, Rokhsar Daniel S.

Science 298 （ 5601 ） 2157 - 2167 2002年12月

ISSN 0036-8075

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains ̃16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

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Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

Hotta Kohji, Takahashi Hiroki, Asakura Tomomi, Saitoh Banjo, Takatori Naohito, Satou Yutaka, Satoh Nori

Developmental Biology 224 （ 1 ） 69 - 80 2000年08月

ISSN 0012-1606

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, β4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos. (C) 2000 Academic Press.

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Brachyury downstream notochord differentiation in the ascidian embryo

Takahashi Hiroki, Hotta Kohji, Erives Albert, Di Gregorio Anna, Zeller Robert W., Levine Michael, Satoh Nori

Genes and Development 13 （ 12 ） 1519 - 1523 1999年06月

ISSN 0890-9369

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation.

Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo

Hotta Kohji, Takahashi Hiroki, Erives Albert, Levine Michael, Satoh Nori

Development Growth and Differentiation 41 （ 6 ） 657 - 664 1999年

ISSN 0012-1592

Expression of the Brachyury (Ci-Bra) gene of the ascidian Ciona intestinalis is initiated at the 64-cell stage. Gene expression is restricted to notochord precursor cells, and Ci-Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci-Bra-downstream genes that are expressed in notochord cells were isolated. The present determination, by whole-mount in situ hybridization, of the temporal expression patterns of 19 notochord-specific and 20 notochord-predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord- specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

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Repression of an ascidian gene in the tip of the tail of tail-bud-stage embryos

Hotta Kohji, Takahashi Hiroki, Satoh Noriyuki

Development Genes and Evolution 208 （ 3 ） 164 - 167 1998年

ISSN 0949-944X

The occurrence of tadpole-type larvae is one of the key events used to understand the origin and evolution of chordates, and the rail bud of chordate embryos is thought to be associated with formation of the tip of the tail. Although some transcriptional factor genes including Brachyury are expressed in the tail bud, no structural genes have been reported to be expressed there. We report here that an ascidian gene HrTT-1 is expressed exclusively in the tip of elongating tail of the tail-bud embryo. This gene encodes a possible secreted protein of 415 amino acids with the SCP/TPX family consensus sequences.

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堀田　耕司

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