Language：English   Publisher：The Biophysical Society of Japan  

<p>Singularity biology is a scientific field that targets drastic state changes in multicellular systems, aiming to discover the key cells that induce the state change and investigate the mechanisms behind them. To achieve this goal, we developed a trans-scale optical imaging system (trans-scale scope), that is capable of capturing both macroscale changes across the entire system and the micro-scale behavior of individual cells, surpassing the cell observation capabilities of traditional microscopes. We developed two units of the trans-scale scope, named AMATERAS-1 and -2, which demonstrated the ability to observe multicellular systems consisting of over one million cells in a single field of view with sub-cellular resolution. This flagship instrument has been used to observe the dynamics of various cell species, with the advantage of being able to observe a large number of cells, allowing the detection and analysis of rare events and cells such as leader cells in multicellular pattern formation and cells that spontaneously initiate calcium waves. In this paper, we present the design concept of AMATERAS, the optical configuration, and several examples of observations, and demonstrate how the strength-in-numbers works in life sciences.</p>

DOI： 10.2142/biophysico.bppb-v21.s017

Web of Science

Scopus

PubMed

CiNii Research

Publisher：Development (Cambridge)  

DOI： 10.1242/dev.202031

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi.org/10.3389/fphys.2022.893736

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/dev.199999

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s42003-019-0336-3

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1159/000477452

Language：English   Publishing type：Research paper (scientific journal)  

The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling-mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown. Here, we report that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons inmice. Analysis ofmice with an inducible Sall1 deletion revealed that Sall1 activates genes expressed in progenitors while repressing genes expressed in differentiating nephrons. Sall1 and Six2 co-occupied many progenitor-related gene loci, and Sall1 bound to Six2 biochemically. In contrast, Sall1 did not bind to the Wnt4 locus suppressed by Six2. Sall1-mediated repressionwas also independent of its binding to DNA. Thus, Sall1maintains nephron progenitors and their derivatives by a unique mechanism, which partly overlaps but is distinct fromthat of Six2: Sall1 activates progenitor-related genes in Six2-positive nephron progenitors and represses gene expression in Six2-negative differentiating nascent nephrons.

DOI： 10.1681/ASN.2013080896

Language：English   Publishing type：Research paper (scientific journal)  

During the early formation of the dorsal aorta, the first-forming embryonic vessel in amniotes, a subset of somitic cells selected as presumptive angioblasts, migrates toward the dorsal aorta, where they eventually differentiate into endothelial cells. We have recently shown that these processes are controlled by Notch signals (Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S., Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y., 2008. Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos. Dev. Cell 14, 890-901.). Here, we studied a possible link between Notch and chemokine signals, SDF1/CXCR4, the latter found to be dominantly expressed in developing aorta/somites. Although CXCR4 overexpression caused a directed migration of somitic cells to the aortic region in a manner similar to Notch, no positive epistatic relationships between Notch and SDF1/CXCR4 were detected. After reaching the aortic region, the CXCR4-electroporated cells exhibited no endothelial character. Importantly, however, once provided with Notch activity, they could successfully be incorporated into developing vessels as endothelial cells. These findings were obtained combining the tetracycline-inducible gene expression method with the transposon-mediated stable gene transfer technique. We conclude that Notch activation is sufficient to direct naïve mesenchymal cells to differentiate into endothelial cells once the cells are conveyed to the aortic region.

DOI： 10.1016/j.ydbio.2009.08.010

Language：English   Publishing type：Research paper (scientific journal)  

During early morphogenesis, tissue segregation is often accompanied by changes in cell shape. To understand how such coordination is regulated, somitogenesis was used as a model. When a somite forms in the anterior end of the presomitic mesoderm, an intersomitic boundary (gap) emerges, and it is rapidly followed by cell epithelialization at this border. It has been known that the gap formation is regulated by intercellular signals. We here demonstrate that cMeso-1, the chicken homolog of mouse Mesp2, upregulates EphA4 in the cells located posteriorly to a forming boundary. This in turn activates EphrinB2-reverse signals in the anteriorly juxtaposed cells, where the EphrinB2 signal is sufficient to cause a gap formation and cell epithelialization cell-autonomously. During these processes, Cdc42 needs to be repressed via tyrosine phosphorylation of EphrinB2. This is the first demonstration that Ephrin-reverse signal acts as a platform that couples distinct morphogenetic changes in cell polarity and tissue shape.

DOI： 10.1073/pnas.0902859106

Language：English   Publishing type：Research paper (scientific journal)  

Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.

DOI： 10.1016/j.ydbio.2005.12.001

Language：English   Publishing type：Research paper (scientific journal)  

We describe here a novel inductive action that operates during somitic segmentation in chicken embryos. We previously reported that the posterior border cells located at a next-forming boundary in the anterior end of the presomitic mesoderm (PSM) exhibit an inductive activity that acts on the anterior cells to cause the formation of a somitic fissure (Sato, Y., Yasuda, K., Takahashi, Y., 2002. Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation. Development 129, 3633-3644). In this study, we have found a second inductive action along the dorso-ventral (D-V) axis during fissure formation. When relocated into a non-segmenting region of PSM, the ventral-most cells taken from the presumptive boundary are sufficient to induce an ectopic fissure in host cells. The ventrally derived signal acts in a ventral-to-dorsal direction but not ventrally, regardless of where the ventral cells are placed. This directional signaling is governed, at least in part, by the signal-receiving cells of the PSM, which we found to be polarized along the D-V axis, and also by intimate cell-cell interactions. Finally, we have observed that morphological segmentation is able to rearrange the anterior and posterior regionalization of individual somites. These findings suggest that discrete unidirectional signals along both the antero-posterior and the D-V axes act coordinately to achieve the formation of the intersomitic fissure, and also that fissure formation is important for the fine-tuning of A-P regionalization in individual somites.

DOI： 10.1016/j.ydbio.2005.03.007

Language：English   Publishing type：Research paper (scientific journal)  

In the Pax gene family, which encodes DNA-binding proteins, Pax 2 has been known to play important roles in the formation of the midbrain/hindbrain boundary, eye, inner ear and kidney in vertebrates (Bioessays 19 (1997) 755). In this article, we report a segmentally regulated pattern of Pax 2 expression during chicken somitogenesis. Pax 2 mRNA is localized in the rostral end of the unsegmented presomitic mesoderm (PSM), abutting anteriorly on a prospective segmentation border. This pattern repeats every segmentation cycle (90 min) observed in ovo and also in the hall embryo culture assay in which one hall of PSM along the midline is fixed immediately while the other half is cultured for a given period. We also determined the sequence of changes in Pax 2 expression during a segmentation cycle by comparing the pattern of Pax 2 with that of Lunatic-fringe (L-fringe), known to cycle periodically in posterior PSM. A systematic comparison of the expression patterns between Pax 2, L-fringe and EphA4 further highlighted a close relationship between EphA4 and Pax 2 during a segmentation cycle. Lastly, Pax 2 is not segmentally expressed in mouse PSM, suggestive of species (avian)-specific mechanisms underlying somitic segmentation.

DOI： 10.1016/S0925-4773(03)00109-6

Language：English   Publishing type：Research paper (scientific journal)  

In the Pax gene family, which encodes DNA-binding proteins, Pax 2 has been known to play important roles in the formation of the midbrain/hindbrain boundary, eye, inner ear and kidney in vertebrates (Bioessays 19 (1997) 755). In this article, we report a segmentally regulated pattern of Pax 2 expression during chicken somitogenesis. Pax 2 mRNA is localized in the rostral end of the unsegmented presomitic mesoderm (PSM), abutting anteriorly on a prospective segmentation border. This pattern repeats every segmentation cycle (90 min) observed in ovo and also in the half embryo culture assay in which one half of PSM along the midline is fixed immediately while the other half is cultured for a given period. We also determined the sequence of changes in Pax 2 expression during a segmentation cycle by comparing the pattern of Pax 2 with that of Lunatic-fringe (L-fringe), known to cycle periodically in posterior PSM. A systematic comparison of the expression patterns between Pax 2, L-fringe and EphA4 further highlighted a close relationship between EphA4 and Pax 2 during a segmentation cycle. Lastly, Pax 2 is not segmentally expressed in mouse PSM, suggestive of species (avian)-specific mechanisms underlying somitic segmentation.

DOI： 10.1016/S0925-4773(02)00344-1

Language：English   Publishing type：Research paper (scientific journal)  

Boundary formation plays a central role in differentiating the flanking regions that give rise to discrete tissues and organs during early development. We have studied mechanisms by which a morphological boundary and tissue separation are regulated by examining chicken somite segmentation as a model system. By transplanting a small group of cells taken from a presumptive border into a non-segmentation site, we have found a novel inductive event where posteriorly juxtaposed cells to the next-forming border instruct the anterior cells to become separated and epithelialized. We have further studied the molecular mechanisms underlying these interactions by focusing on Lunatic fringe, a modulator of Notch signaling, which is expressed in the region of the presumptive boundary. By combining DNA in ovo electroporation and embryonic transplantation techniques we have ectopically made a sharp boundary of Lunatic fringe activity in the unsegmented paraxial mesoderm and observed a fissure formed at the interface. In addition, a constitutive active form of Notch mimics this instructive phenomenon. These suggest that the boundary-forming signals emanating from the posterior border cells are mediated by Notch, the action of which is confined to the border region by Lunatic fringe within the area where mRNAs of Notch and its ligand are broadly expressed in the presomitic mesoderm.

Event date： 2016.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Tsukuba International Conference Center   Country：Japan  

Event date： 2015.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：Kobe International Conference Center   Country：Japan  

Event date： 2015.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Shaanxi Guesthouse Hotel, Xi’an   Country：China  

Event date： 2014.11

Language：Japanese  

Country：Japan  

Event date： 2023.11 - 2023.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：幕張メッセ   Country：Japan  

血管内皮細胞は分化した場所から長距離を移動し、心臓を中心とした広域ネットワークを作りだす。器官レベルにおける血管ネットワークパターンは多様性に富み、各臓器が果たす生理代謝調節機能や疾患時の病態に直結している。ミクロからマクロレベルまで連続的階層性を有する血管パターニング制御機構の解明は、医学的・生物学的に重要な課題である。従来型の顕微鏡システムを用いた生体イメージング研究から、特に末梢部の血管形成機構は飛躍的に解明が進んだ。一方で一括イメージングできる範囲が限られてきたことから、全身性の広域ネットワークがどのようにして確立されるのかは十分に理解されていない。
　鳥類胚は胚盤上に広大な血管ネットワークを発達させるため、血管形成研究の主要なモデル実験系として多くの発見を導いてきた。他のモデル動物にはない鳥類胚の最大の特徴は、35 mmディッシュ内で胚盤全体を含むホールマウント培養が可能な点である。この特徴を生かして血管形成過程を詳細にするため、我々は血管内皮細胞の核を可視化できるトランスジェニックウズラtie1:H2B-eYFP胚に対し、近年新たに開発されたトランススケール顕微鏡AMATERAS（Ichimura et al., Sci Rep, 2021)によるタイムラプス観察解析を行っている。本ワークショップでは、広範囲・高精細イメージングを可能にするAMATERAS顕微鏡によってどのような細胞挙動をとらえることができるのか、さらに画像データ解析から血管形成期における特異点を探索する試みを紹介する。

Event date： 2022.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：パシフィコ横浜   Country：Japan  

Event date： 2019.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Iwate University   Country：Japan  

Event date： 2018.6 - 2019.6

Language：English  

Venue：Tower Hall Funabori   Country：Japan  

Event date： 2018.6

Language：English  

Venue：Tower Hall Funabori   Country：Japan  

Event date： 2018.1 - 2019.1

Language：English  

Venue：Kumamoto City International Center   Country：Japan  

Event date： 2017.12 - 2019.5

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：奈良先端科学技術大学院大学   Country：Japan  

Event date： 2015.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：ホテルメトロポリタンエドモント飯田橋   Country：Japan  

Event date： 2015.6

Language：English  

Venue：International Congress Center, Tsukuba   Country：Japan  

Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

Venue：Unitopia Sasayama   Country：Japan  

Language：English  

During early development in vertebrates, cells change their shapes dramatically both from epithelial to mesenchymal and also from mesenchymal to epithelial, enabling the body to form complex tissues and organs. Using somitogenesis as a novel model, Rho family GTPases have recently been shown to play essential and differential roles in individual cell behaviors in actual developing embryos. Levels of Cdc42 activity provide a binary switch wherein high Cdc42 levels allow the cells to remain mesenchymal, while low Cdc42 levels produce epithelialization. Rac1 activity needs to be precisely controlled for proper epithelialization through the bHLH transcription factor Paraxis. Somitogenesis is expected to serve as an excellent model with which one can understand how the functions of developmental genes are resolved into the morphogenetic behavior of individual cells.

DOI： 10.1159/000084507