1. Yamamoto, M., Ohsawa, S., Kunimasa K., and Igaki T.
The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition.
Nature, 542, 246-250 (2017).
DOI: 10.1038/nature21033

Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Here, through a genetic screen in Drosophila, we identify the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition.


(q)正常細胞のSasが極性崩壊細胞のPTP10Dに結合すると、極性崩壊細胞内のEGF受容体(EGFR)によるRasの活性化が抑制される。これにより、Eiger-JNKシグナルが極性崩壊細胞に対して細胞死を誘導する。(r) Sas-PTP10Dの結合が起こらない場合には、極性崩壊細胞内でEGFR-Rasシグナルが活性化し、Eiger-JNKシグナルを「細胞死」から「F-actinの蓄積・Ykiの活性化を介した細胞増殖」シグナルへと変換する。これにより、極性崩壊細胞の排除が抑制され、極性崩壊細胞の過剰な増殖と腫瘍化が引き起こされる。


1. Inaki, M., Yang L. J., and Matsuno K.
Left-right asymmetric morphogenesis in Drosophila and other invertebrates: the discovery of intrinsic cell chirality and its functions.
Reviews in Cell Biology and Molecular Medicine in press (2017)

The formation of left-right (LR) asymmetry is one of the fundamental problems to be solved in developmental biology. In various vertebrate species, the LR axis forms as a result of a leftward flow of extraembryonic fluid that is generated by motile cilia. However, recent studies show that the mechanisms of LR-asymmetric development are evolutionarily divergent even among vertebrates. In snails and nematodes, the LR asymmetry of blastomeres plays a key role in the LR-asymmetric disposition of blastomeres during subsequent development, leading to LR-asymmetric cell-cell interactions among the blastomeres. Such LR asymmetry of cells can be defined as cell chirality. An object has chirality if it cannot be superimposed onto its mirror image. In some Drosophila organs, epithelial cells have intrinsic cell chirality that drives LR-asymmetric morphogenesis. Thus, the mechanisms of LR-asymmetric development in Lophotrochozoa and Ecdysozoa differ from the motile-cilia-driven mechanisms found in vertebrates. Although intrinsic cell chirality has been observed in various cultured vertebrate cells, the biological role of this chirality is unknown. Cell chirality might be a general mechanism for LR-asymmetric development across phyla.




1. Maeda, E., Nakagaki, M., Ichikawa, K., Nagayama, K., and Matsumoto,
T. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.
Bone Reports 6, 120-8 (2017).
DOI: 10.1016/j.bonr.2017.04.002.

Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important information to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to the loading direction. The ex vivo loading system established here can be further applied to study responses to mechanical loading in osteogenesis as well as callus for better understanding of factors to consider in successful mechanically inducing bone regeneration.





1. Shinobu Hirai, Kohji Hotta, Yoshihiro Kubo, Atsuo Nishino, Shigeo Okabe, Yasushi Okamura, and Haruo Okado
AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.
PNAS, accepted

AMPA-type glutamate receptors (GluAs) mediate fast excitatory transmission in the vertebrate central nervous system (CNS), and their function has been extensively studied in the mature mammalian brain. However, GluA expression begins very early in developing embryos, suggesting that they may also have unidentified developmental roles. Here, we identify developmental roles for GluAs in the ascidian Ciona intestinalis. Mammals express Ca2+-permeable GluAs (Ca-P GluAs) and Ca2+-impermeable GluAs (Ca-I GluAs) by combining subunits derived from four genes. In contrast, ascidians have a single gluA gene. Taking advantage of the simple genomic GluA organization in ascidians, we knocked down (KD) GluAs in Ciona and observed severe impairments in formation of the ocellus, a photoreceptive organ used during the swimming stage, and in resorption of the tail and body axis rotation during metamorphosis to the adult stage. These defects could be rescued by injection of KD-resistant GluAs. GluA KD phenotypes could also be reproduced by expressing a GluA mutant that dominantly inhibits glutamate-evoked currents. These results suggest that, in addition to their role in synaptic communication in mature animals, GluAs also have critical developmental functions.




1. Matsuo I., and Hiramatsu R.
Mechanical perspectives on the anterior-posterior axis polarization of mouse implanted embryos.
Mech Dev. 144(Pt A): 62-70 (2017)
DOI: 10.1016/j.mod.2016.09.002.

In most mammals, embryonic development and growth proceed in the maternal uterus. Mouse late blastocyst embryos implant on the uterine epithelium around embryonic day (E)4.5, and immediately afterward the whole embryo's shape is dynamically changed from a bowl-like shape to an elongated egg-cylinder until E5.5. Concurrently, mouse anterior-posterior (A-P) axis polarization occurs by the emergence of distal visceral endoderm (DVE) scells at the cellular and molecular levels as the proximal-distal (P-D) axis. The embryonic growth and axis polarization are considered to be controlled primarily by multiple growth factors' signaling. However, the precise cellular mechanisms of DVE formation in which this signaling is involved have been unclear. We recently identified that local breaching of the basement membrane (BM) between the epiblast and the visceral endoderm (VE) at the distal tip allows inner epiblast cells to transmigrate into the outer VE layer as the emergence of DVE cells. More importantly, the local BM loss in the distal region appears to be triggered by mechanical forces exerted from maternal tissues on embryos and embryonic growth itself. Our data suggest a fascinating hypothesis concerning mouse A-P axis polarization mediated by the whole embryo's shape change through mechanical stress between the embryo and the uterine epithelium. Our mechanical model provides a unique insight into why the first axis polarity of the implanted mouse embryo is established in the P-D direction initially and not in the future A-P direction. We also discuss whether the local breaching of the BM mediated by mechanical cues is essential to mouse A-P axis polarization in in vitro culture.

マウス胚の形状は、着床後お椀形から卵筒形にダイナミックに変化する。その過程で、円筒形の遠位側先端部分で将来の前方となる細胞が出現し、前後軸が形成されるが、なぜ円筒形になることと前後軸形成との関係は、長い間不明であった。我々は、エピブラストと臓側内胚葉の間に存在する基底膜が切れて、内側のエピブラスト細胞が外側の臓側内胚葉層に飛び出すことを見いだした。更に、この基底膜切断は、子宮内膜組織から胚へかかる圧力と胚自身の成長に依存していることを明らかにした。本総説では、機械的な要因で基底膜が局所的に切断することが前後軸極性化に必須なステップであるかどうか、更にはin vitroマウス胚培養下での前後軸極性化においても妥当性があるかどうかについて議論している。

基底膜の沈着分布と遠位臓側内胚葉(DVE, 前後軸)形成
(A)子宮内や円筒形のPDMSデバイス内の胚では、基底膜は遠位(distal)側ほど減少し、一定量(Threshold for DVE formation)以下になると基底膜切断が起こり、遠位臓側内胚葉(DVE、前後軸)が形成される。(B)コラ−ゲナーゼ処理後の胚では、基底膜の沈着は劇的に減少し、より近位側でも一定量以下になるため、DVEがより広範囲に形成される。(C)成長しないSmad4欠損胚や阻害剤によって成長が抑制された胚では、遠位方向に胚が伸長しないため基底膜の沈着が一定量以下にならないため、DVEは形成されない。(D)浮遊培養下で、長期間培養すると、胚は、正常条件より大きく伸長することができるため、基底膜の沈着が一定量以下になる領域が出現する。その結果、お椀形でもDVEが形成される。




1. Nishikawa, S., Takamatsu, A., Ohsawa, S., and Igaki, T.
Mathematical model for cell competition: Predator-prey interactions at the interface between two groups of cells in monolayer tissue.
J. Theor. Biol., 404, 40-50 (2016).
DOI: 10.1016/j.jtbi.2016.05.031

The phenomenon of 'cell competition' has been implicated in the normal development and maintenance of organs, such as in the regulation of organ size and suppression of neoplastic development. In cell competition, one group of cells competes with another group through an interaction at their interface. Which cell group "wins" is governed by a certain relative fitness within the cells. However, this idea of cellular fitness has not been clearly defined. We construct two types of mathematical models to describe this phenomenon of cell competition by considering the interaction at the interface as a predator-prey type interaction in a monolayer tissue such as epithelium. Both of these models can reproduce several typical experimental observations involving systems of mutant cells (losers) and normal cells (winners). By analyzing one of the model and defining an index for the degree of fitness in groups of cells, we show that the fate of each group mainly depends on the relative carrying capacities of certain resources and the strength of the predator-prey interaction at the interface. This contradicts the classical hypothesis in which the relative proliferation rate determines the winner.

多細胞コミュニティーにおいて隣り合う細胞同士がその生存を競い合う「細胞競合」は正常発生や恒常性維持に重要な役割を果たすことが知られているが、競合の“勝者”“敗者”を規定する「組織への適応度」の実体はいまだ不明である。本研究では、細胞競合における適応度について、数理モデルによる解釈を試みた。2種の集団間の競合モデルとして知られる生態系モデルLotka-Vortella モデルに、境界で生じる「捕食者—被捕食者」型の相互作用を導入し、数学的解析を行った結果、“勝者”“敗者”の細胞運命が、主には2種集団の環境収容力(単独での増殖時の組織サイズ)の比と境界上での細胞間相互作用の強さに依存することが明らかとなった。



1. Inaki, M., Yang L. J., and Matsuno K.
Cell chirality: its origin and roles in left-right asymmetric development.
Phil Trans B 371, 20150403 (2016).
DOI: 10.1098/rstb.2015.0403

An item is chiral if it cannot be superimposed on its mirror image. Most biological molecules are chiral. The homochirality of amino acids ensures that proteins are chiral, which is essential for their functions. Chirality also occurs at the whole-cell level, which was first studied mostly in ciliates, single-celled protozoans. Ciliates show chirality in their cortical structures, which is not determined by genetics, but by 'cortical inheritance'. These studies suggested that molecular chirality directs whole-cell chirality. Intriguingly, chirality in cellular structures and functions is also found in metazoans. In Drosophila, intrinsic cell chirality is observed in various left–right (LR) asymmetric tissues, and appears to be responsible for their LR asymmetric morphogenesis. In other invertebrates, such as snails and DCaenorhabditis elegans, blastomere chirality is responsible for subsequent LR asymmetric development. Various cultured cells of vertebrates also show intrinsic chirality in their cellular behaviours and intracellular structural dynamics. Thus, cell chirality may be a general property of eukaryotic cells. In Drosophila, cell chirality drives the LR asymmetric development of individual organs, without establishing the LR axis of the whole embryo. Considering that organ-intrinsic LR asymmetry is also reported in vertebrates, this mechanism may contribute to LR asymmetric development across phyla.



2. Matsumoto, K., Ayukawa, T., Ishio, A., Sasamura, T., Yamakawa, T. and Matsuno, K.
Dual roles of O-glucose glycans redundant with monosaccharide O-fucose on Notch in Notch Trafficking.
J. Biol. Chem. 291, 13743-13752 (2016).
DOI: 10.1074/jbc.M115.710483.

Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions.




1. Wang, J., Ito, M., Zhong, W., Sugita, S., Michiue, T., Tsuboi, T., Kitaguchi, T., and Matsumoto, T. Observations of intracellular tension dynamics of MC3T3-E1 cells during substrate adhesion using a FRET-based actinin tension sensor.
Journal of Biomechanical Science and Engineering
11, 16-00504 (2016).
DOI: 10.1299/jbse.16-00504.

Tension in actin filaments plays crucial roles in multiple cellular functions, although little is known about tension dynamics of cells during adhesion to substrates. In this study, we visualized intracellular tension in actin filaments using a newly developed Förster resonance energy transfer (FRET)-based tension sensor (Actinin-sstFRET-GR). Tension dynamics were monitored during adhesion in MC3T3-E1 mouse osteoblastic cells after introduction of the sensor. Whole cell areas continued to increase from 10 to 180 min after plating and tension was monitored in a typical section close to the bottom, where the mean fluorescence was the largest. Tension on the bottom side was negatively correlated with cell area from 10 to 110 min. Thereafter, this correlation was positive until 180 min. We then analyzed central-peripheral differences in tension close to the bottom. In these experiments, tension in the cell periphery increased during expansion of the area and decreased during contraction. As a result, fluctuations of tension in this area were much larger than those in the central area of the cell. Finally, we analyzed upper-lower differences in tension development during initial adhesion and showed continuous decreases in the lower side of the cell from 10 to 110 min after plating, and decreases in the upper side until 70 min, followed by increases. In this study, we successfully visualized dynamic changes in intracellular tension at the sub-cellular level and found that development of tension during initial adhesion processes is time-dependent and has central-peripheral and upper–lower differences. The present FRET tension sensor may become a powerful tool in studies of cell biomechanics.


FRET張力センサを発現したMC3T3-E1細胞の基板接着に伴う張力変化の様子。接着するに連れ、アクチンに作用する張力が減少(FRET ratioが増加)していること判る。また細胞の突出部(*)周辺の張力が高いことも判る。

2. Wang, J.F., Sugita, S., Nagayama, K., Matsumoto, T.
Dynamics of actin filaments of MC3T3-E1 cells during adhesion process to substrate.
Journal of Biomechanical Science and Engineering 11, 15-00637 (2016).
DOI: 10.1299/jbse.15-00637.

In order to determine how cells change their morphology during adhesion process to a substrate, we focused on the actin cytoskeleton and investigated its morphological change along with that of the whole cell during adhesion process. An osteoblastic cell line MC3T3-E1 was used as the test model. We plated cells whose cell cycle had been synchronized by serum starvation on fibronectin-coated glass plate and cultured them for 10 min to 24 h. We then stained their F-actin and nucleus and observed them with a fluorescent microscope for cell area and shape index and 2D parameters for actin morphology, and with a laser scanning microscope for 3D morphology of actin and nucleus. In the beginning of adhesion, the trypsinized cells were round and their nuclei were surrounded uniformly by thick layer of actin. The actin layer in the upper side became actin aggregate (AA) and lower side dense peripheral band (DPB) in 30 min. The upper AA then became smaller and finally to actin filaments (AFs) spanning the cell top. The DPB expanded and finally became AFs on cell bottom by 1 h. The nucleus becomes flattened possibly due to compression by the cell membrane caused by the expansion of the DPB in the early stage of adhesion. In the later stage of adhesion, the number of AFs continuously increased and nucleus became flattened more and more until 12 h. This may be caused by the increase in the top AFs that may compress the nucleus. Cells become more elongated in response to further alignment of AFs until 12 h. These results indicate that change in AFs during adhesion process is complicated not only temporally but also spatially.

芽細胞様細胞MC3T3-E1の基板接着時のアクチンフィラメントの動態を細かく観察した。トリプシンで剥離された細胞は最初、厚いアクチンの層で覆われているが、基板に触れると、基板と核の間のアクチンが分解され、周辺のアクチンがdense peripheral bandに、上部のアクチンがstress fiberに変化するらしいことが示された。




1. Maryu, G., Matsuda, M., and Aoki, K.
Multiplexed fluorescence imaging of ERK and Akt activities and cell-cycle progression.
Cell Struct.Funct., 41, 41:81-92 (2016).
DOI: 10.1247/csf.16007.

The Ras-ERK pathway controls cell proliferation and differentiation, whereas the PI3K-Akt pathway plays a role in the process of cell-cycle progression and cell survival. Both pathways are activated by many stimuli such as epidermal growth factor (EGF), and coordinately regulate each other through cross-talk. However, it remains unclear how cells accommodate the dynamics and interplay between the Ras-ERK and PI3K-Akt pathways to regulate cell-fate decisions, mainly because of the lack of good tools to visualize ERK and Akt activities simultaneously in live cells. Here, we developed a multiplexed fluorescence system for imaging ERK and Akt signaling and the cell-cycle status at the single cell level. Based on the principle of the kinase translocation reporter (KTR), we created Akt-FoxO3a-KTR, which shuttled between nucleus and cytoplasm in a manner regulated by Akt phosphorylation. To simultaneously measure ERK, Akt and the cell-cycle status, we generated a polycistronic vector expressing ERK-KTR, Akt-FoxO3a-KTR, a cell-cycle reporter and a nuclear reporter, and applied linear unmixing to these four images to remove spectral overlap among fluorescent proteins. The specificity and sensitivity of ERK-KTR and Akt-FoxO3a-KTR were characterized quantitatively. We examined the cellular heterogeneity of relationship between ERK and Akt activities under a basal or EGF-stimulated condition, and found that ERK and Akt were regulated in a highly cooperative and cell-cycle-dependent manner. Our study provides a useful tool for quantifying the dynamics among ERK and Akt activities and the cell cycle in a live cell, and for addressing the mechanisms underlying intrinsic resistance to molecularly targeted drugs.




1. Sakai, Y., Tachikawa, M. and Mochizuki, A.
Controlling segregation speed of entangled polymers by the shapes: A simple model for eukaryotic chromosome segregation.
Phys. Rev. E 94, 042403 (2016).
DOI: 10.1103/PhysRevE.94.072403.

We report molecular dynamics simulations of the segregation of two overlapping polymers motivated by chromosome segregation in biological cells. We investigate the relationship between polymer shapes and segregation dynamics and show that elongation and compaction make entangled polymers segregate rapidly. This result suggests that eukaryotic chromosomes take such a characteristic rod-shaped structure, which is induced by condensins, to achieve rapid segregation.




1. Tanigawa S, Taguchi A, Sharma N, Perantoni AO, and Nishinakamura R.
Selective in vitro propagation of nephron progenitors from embryos and pluripotent stem cells.
Cell Reports 15, 801-813 (2016).
DOI: 10.1016/j.celrep.2016.03.076

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine.

腎臓はネフロンと呼ばれる機能単位の集合体である。ネフロンは胎児期にネフロン前駆細胞から作られるが、このネフロン前駆細胞は出生前後に消失してしまい、それが腎臓が再生しない一因とされている。 今回我々は、LIF, WNT及びBMPを敢えて低い濃度で添加することによって、マウスの胎仔から単離したネフロン前駆細胞を試験管内で約20日間培養し、約1,800倍に増幅することに成功した。この培養法をヒトiPS細胞から誘導したネフロン前駆細胞に適用したところ、8日間で4倍に増幅することができた。本研究はネフロン前駆細胞を人為的に増幅するもので、腎疾患の病態解明、創薬及び細胞治療など大量に細胞を必要とする再生医療に向けた大きな一歩である。


2. Sharmin S, Taguchi A, Kaku Y, Yoshimura Y, Ohmori T, Sakuma T, Mukoyama M, Yamamoto T, Kurihara H and Nishinakamura R.
Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation.
Journal of the American Society of Nephrology 27, 1778-1791 (2016).
DOI: 10.1681/ASN.2015010096

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.


A. 試験管内で作製した緑に光るヒト腎臓組織。丸い一つ一つが糸球体。
B. マウスへの移植によって血管を取り込んだヒトiPS細胞由来の糸球体。
C. マウス血管(緑色)がポドサイト(桃色)の間に入り込んでいる。
D. ポドサイトの細胞突起間に形成されたろ過膜構造(矢印)


1. Nishihara H., Kobayashi N., Kimura-Yoshida C., Yan K., Bormuth O., Ding Q., Nakanishi A., Sasaki T., Hirakawa M., Sumiyama K., Furuta Y., Tarabykin V., Matsuo I., and Okada N.
Coordinately Co-opted Multiple Transposable Elements Constitute an Enhancer for wnt5a Expression in the Mammalian Secondary Palate.
PLoS Genet. 12(10):e1006380. (2016)
DOI: 10.1371/journal.pgen.1006380.

Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.






1. Enomoto, M., Kizawa, D., Ohsawa, S., and Igaki, T.
JNK signaling is coverted from anti- to pro-tumor pathway by Ras-mediated switch of Warts activity. Dev. Biol., 403, 162-171 (2015).
DOI: 10.1016/j.ydbio.2015.05.001

The c-Jun N-terminal kinase (JNK) pathway is a dual-functional oncogenic signaling that exerts both anti- and pro-tumor activities. However, the mechanism by which JNK switches its oncogenic roles depending on different cellular contexts has been elusive. Here, using the Drosophila genetics, we show that hyperactive Ras acts as a signaling switch that converts JNK's role from anti- to pro-tumor signaling through the regulation of Hippo signaling activity. In the normal epithelium, JNK signaling antagonizes the Hippo pathway effector Yorkie (Yki) through elevation of Warts activity, thereby suppressing tissue growth. In contrast, in the presence of hyperactive Ras, JNK signaling enhances Yki activation by accumulating F-actin through the activity of the LIM domain protein Ajuba, thereby promoting tissue growth. We also find that the epidermal growth factor receptor (EGFR) signaling uses this Ras-mediated conversion of JNK signaling to promote tissue growth. Our observations suggest that Ras-mediated switch of the JNK pathway from anti- to pro-tumor signaling could play crucial roles in tumorigenesis as well as in normal development.




1. Otsuka, T., Tsukahara, T., Takeda, H.
Development of the pancreas in medaka, Oryzias latipes, from embryo to adult.
Dev Growth Differ. 57, 557-569 (2015).
DOI: 10.1111/dgd.12237.

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1- and ptf1a-transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.



1. Imai, M., Furusawa, K., Mizutani, T., Kawabata, K. and Haga, H. Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate.
Scientific Reports. 5, 14208, 1-10 (2015).
DOI: 10.1038/srep14208.

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.



2. Takemoto, K., Ishihara, S., Mizutani, T., Kawabata, K. and Haga, H.
Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway.
PLOS ONE. 10, e0117937 (2015).
DOI: 10.1371/journal.pone.0117937.

Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.



3. Yamaguchi, N., Mizutani, T., Kawabata, K. and Haga, H.
Leader Cells Regulate Collective Cell Migration via Rac Activation in the Downstream Signaling of Integrin Beta1 and PI3K.
Scientific Reports. 5, 7656, 1-8 (2015).
DOI: 10.1038/srep07656.

Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.




1. Okumura, T., Sasamura, T., Inatomi, M., Hozumi, S., Nakamura, M., Hatori, R., Taniguchi, K., Nakazawa,N., Suzuki, E., Maeda, R., Yamakawa, T., and Matsuno, K.
Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila.
Genetics 199 (4) 1183-1199 (2015).
DOI: 10.1534/genetics.115.174698.

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.



2. Ishio, A., Sasamura, T., Ayukawa, T., Kuroda, J., Ishikawa, H. O., Aoyama, N., Matsumoto, K., Gushiken, T., Okajima, T., Yamakawa, T., and Matsuno, K.
O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation.
J. Biol. Chem. 290, 505-519 (2015).
DOI: 10.1074/jbc.M114.616847.

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1(R245A knock-in)), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1(R245A knock-in) and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein's functions.

Notchシグナルは、ショウジョウバエの左右非対称性形成に必須である。Notchシグナルは、細胞間相互作用を介した細胞運命決定において重要な役割を担っている。膜貫通型受容体であるNotchの細胞外ドメインには、リガンドが結合するEGF様リピート存在する。これらのEGF様リピートの一部には、protein O-fucosyltransferase 1(O-fut1)によってO-フコースが、RumiによってO-グルコースが付加される。我々は、O-fut1遺伝子がNotchシグナル伝達に必須であることを明らかにした。一方、O-fut1は、酵素活性非依存的な、Notchに対するシャペロン機能をもっている。O-fut1遺伝子がNotchシグナルに必須であることは、O-fut1のシャペロン機能がNotchシグナルに不可欠であると考えても説明できることから、NotchのO-フコース単糖修飾の機能の有無については、異なった結果が報告されていた。



1. Nagayama, K., Hamaji, Y., Sato, Y., and Matsumoto, T.
Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells.
J. Biomechanics 48, 1796-1803 (2015).
DOI: 10.1016/j.jbiomech.2015.05.004.

The interaction between cells and the extracellular matrix on a topographically patterned surface can result in changes in cell shape and many cellular functions. In the present study, we demonstrated the mechanical deformation and trapping of the intracellular nucleus using polydimethylsiloxane (PDMS)-based microfabricated substrates with an array of micropillars. We investigated the differential effects of nuclear deformation on the proliferation of healthy vascular smooth muscle cells (SMCs) and cervical cancer HeLa cells. Both types of cell spread normally in the space between micropillars and completely invaded the extracellular microstructures, including parts of their cytoplasm and their nuclei. We found that the proliferation of SMCs but not HeLa cells was dramatically inhibited by cultivation on the micropillar substrates, even though remarkable deformation of nuclei was observed in both types of cells. Mechanical testing with an atomic force microscope and a detailed image analysis with confocal microscopy revealed that SMC nuclei had a thicker nuclear lamina and greater expression of lamin A/C than those of HeLa cells, which consequently increased the elastic modulus of the SMC nuclei and their nuclear mechanical resistance against extracellular microstructures. These results indicate that the inhibition of cell proliferation resulted from deformation of the mature lamin structures, which might be exposed to higher internal stress during nuclear deformation. This nuclear stress-induced inhibition of cell proliferation occurred rarely in cancer cells with deformable nuclei.


正常血管平滑筋細胞(SMCs)とHeLa細胞(HeLa)の培養中の様子(A-H)と基板形態が細胞増殖に与える影響(I, J)。

2. Nakayama, S., Arima, K., Kawai, K., Mohri, K., Inui, C., Sugano, W., Koba, H., Tamada, K., Nakata, Y.J., Kishimoto, K., Arai-Shindo, M., Kojima, C., Matsumoto, T., Fujimori, T., Agata, K., and Funayama, N.
Dynamic transport and cementation of skeletal elements build up pole-and-beam structured skeleton of sponges.
Current Biology 25, 1-6 (2015).
DOI: 10.1016/j.cub.2015.08.023.

Few studies to date have investigated the cellular mechanisms by which sponges develop their characteristic structure. In most sponges, skeletal components known as spicules are connected to form skeletons. For this, a spicule should become appropriately localised in the sponge, one end should be raised up, and the other end becomes fixed to the base of the sponge ("picule holding-up") or to other spicules forming the skeleton ("spicule building-up"). Here we show that novel spicule-carrying cells ("transport cells") are the main builders of the spiculous skeleton. Using a live-imaging technique that we devised to examine the developmental process, we visualised dynamic movement of the spicules used to construct the demosponge skeleton. Transport cells carry spicules to the outer epithelium, which the spicule gradually pierces. The front/outer end of the spicule is then raised up, and its back/basal end becomes fixed, completing one cycle of either spicule holding-up or building-up.





1. Nakayama S, Arima K, Kawai K, Mohri K, Inui C, Sugano W, Koba H, Tamada K, Nakata YJ, Kishimoto K, Arai-Shindo M, Kojima, C, Matsumoto T, Fujimori T, Agata K, and Funayama N.
Dynamic transport and cementation of skeletal elements building up pole-and-beam structured skeleton of sponges
Current Biology 25: 2549-54 (2015).
DOI: 10.1016/j.cub.2015.08.023

Animal bodies are shaped by skeletons, which are built inside the body by biomineralization of condensed mesenchymal cells in vertebrates and echinoderms, or outside the body by apical secretion of extracellular matrices by epidermal cell layers in arthropods. In each case, the skeletons' shapes are a direct reflection of the pattern of skeleton-producing cells. Here we report a newly discovered mode of skeleton-formation: assembly of sponges' mineralized skeletal-elements (spicules) in places distant from where they were produced. Although it was known that internal skeletons of sponges consist of spicules assembled into large pole-and-beam structures with a variety of morphologies, the spicule-assembly-process (i.e., how spicules become held up and connected basically in staggered tandem) and what types of cells act in this process remained unexplored. Here, we discovered that mature spicules are dynamically transported from where they were produced, then pierce through outer epithelia, and their basal ends become fixed to substrate or connected with such fixed spicules. Newly discovered transport cells mediate spicule movement and the pierce step, and collagen-secreting basal-epithelial cells fix spicules to the substratum, suggesting that the processes of spiculous skeleton construction are mediated separately by specialized cells. Division of labor by manufacturer, transporter, and cementer cells, and iteration of the sequential mechanical reactions of "ransport" "pierce" "raise up" and "cementation", allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges.



平成27年度〜31年度 文部科学省科学研究費補助金新学術領域研究(研究領域提案型)生物の3D形態を構築するロジック

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