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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堀田 耕司

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堀田　耕司

論文 - 堀田　耕司