Language：English   Publishing type：Research paper (bulletin of university, research institution)  

All cells, including nonexcitable cells, maintain a discrete transmembrane potential (V mem), and have the capacity to modulate V mem and respond to their own and neighbors' changes in V mem Spatiotemporal variations have been described in developing embryonic tissues and in some cases have been implicated in influencing developmental processes. Yet, how such changes in V mem are converted into intracellular inputs that in turn regulate developmental gene expression and coordinate patterned tissue formation, has remained elusive. Here we document that the V mem of limb mesenchyme switches from a hyperpolarized to depolarized state during early chondrocyte differentiation. This change in V mem increases intracellular Ca2+ signaling through Ca2+ influx, via CaV1.2, 1 of L-type voltage-gated Ca2+ channels (VGCCs). We find that CaV1.2 activity is essential for chondrogenesis in the developing limbs. Pharmacological inhibition by an L-type VGCC specific blocker, or limb-specific deletion of CaV1.2, down-regulates expression of genes essential for chondrocyte differentiation, including Sox9, Col2a1, and Agc1, and thus disturbs proper cartilage formation. The Ca2+-dependent transcription factor NFATc1, which is a known major transducer of intracellular Ca2+ signaling, partly rescues Sox9 expression. These data reveal instructive roles of CaV1.2 in limb development, and more generally expand our understanding of how modulation of membrane potential is used as a mechanism of developmental regulation.

DOI： 10.1073/pnas.1908981116

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

The Müllerian duct (MD) and Wolffian duct (WD) are embryonic tubular tissues giving rise to female and male reproductive tracts, respectively. In amniote embryos, both MD and WD emerge in both sexes, but subsequently degenerate in the males and females, respectively. Here, by using MD-specific gene manipulations in chicken embryos, we identify the molecular and cellular mechanisms that link early MD specification to tubular invagination. Early (pre-)specification of MD precursors in the coelomic epithelium requires BMP signaling and its downstream target Pax2 in a WD-independent process. Subsequently, the BMP/Pax2 axis induces Lim1 expression, a hallmark of MD specification, for which FGF/ERK and WD-derived signals are also required. Finally, the sequential actions of the BMP/Pax2 and FGF/Lim1 axes culminate in epithelial invagination to form a tubular structure driven by an apical constriction, where apical accumulation of phospho-myosin light chain is positively regulated by FGF/ERK signaling. Our study delineates mechanisms governing the early formation of the MD, and also serves as a model of how an epithelial cell sheet is transformed to a tubular structure, a process seen in a variety of developmental contexts.

DOI： 10.1242/dev.137067

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

When a tubular structure forms during early embryogenesis, tubular elongation and lumen formation (epithelialization) proceed simultaneously in a spatiotemporally coordinated manner. We here demonstrate, using the Wolffian duct (WD) of early chicken embryos, that this coordination is regulated by the expression of FGF8, which shifts posteriorly during body axis elongation. FGF8 acts as a chemoattractant on the leader cells of the elongating WD and prevents them from epithelialization, whereas static ('rear') cells that receive progressively less FGF8 undergo epithelialization to form a lumen. Thus, FGF8 acts as a binary switch that distinguishes tubular elongation from lumen formation. The posteriorly shifting FGF8 is also known to regulate somite segmentation, suggesting that multiple types of tissue morphogenesis are coordinately regulated by macroscopic changes in body growth.

DOI： 10.1242/dev.122408

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

Deciphering how the tubulogenesis is regulated is an essential but unsolved issue in developmental biology. Here, using Wolffian duct (WD) formation in chicken embryos, we have developed a novel method that enables gene manipulation during tubulogenesis in vivo. Exploiting that WD arises from a defined site located anteriorly in the embryo (pronephric region), we targeted this region with the enhanced green fluorescent protein (EGFP) gene by the in ovo electroporation technique. EGFP-positive signals were detected in a wide area of elongating WD, where transgenic cells formed an epithelial component in a mosaic manner. Time-lapse live imaging analyses further revealed dynamic behavior of cells during WD elongation: some cells possessed numerous filopodia, and others exhibited cellular tails that repeated elongation and retraction. The retraction of the tail was precisely regulated by Rho activity via actin dynamics. When electroporated with the C3 gene, encoding Rho inhibitor, WD cells failed to contract their tails, resulting in an aberrantly elongated process. We further combined with the Tol2 transposon-mediated gene transfer technique, and could trace EGFP-positive cells at later stages in the ureteric bud sprouting from WD. This is the first demonstration that exogenous gene(s) can directly be introduced into elongating tubular structures in living amniote embryos. This method has opened a way to investigate how a complex tubulogenesis proceeds in higher vertebrates.

DOI： 10.1111/dgd.12047

DOI： 10.1101/2023.06.12.544577

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PLoS Genetics  

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

DOI： 10.1371/journal.pgen.1010614

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Language：English   Publisher：John Wiley & Sons Australia, Ltd  

ニワトリ線維芽細胞および始原生殖細胞(PGC)において、導入遺伝子(EBFP;改変型青色蛍光タンパク質をコードする遺伝子)および内在性遺伝子(cDDX4;DEAD-box型RNAヘリカーゼをコードする遺伝子)に対するプライム編集(PE)について検討した。まず、ニワトリ線維芽細胞を用いてEBFP-EGFP(EGFP;改変型緑色蛍光タンパク質)変換システムを立ち上げた。1塩基の置換でEBFPをEGFPに変換できるため、そのスペクトル変化を利用し、緑色の蛍光を検出することでPEが成功しているかどうかを判断することができた。cDDX4はニワトリPGCの代表マーカーであるため、PGCの編集候補遺伝子として選択した。ニワトリPGCの長期培養系とTol2トランスポゾンを介したゲノム組み込みを活用し、プライム編集PGCのクローンを効率的に得る方法を考案した。本手法により、PGC形成や生殖細胞の発生に関わる分子メカニズムの解明や精密に遺伝子改変されたニワトリの作出への道が開かれるものと考えられた。

Language：English   Publisher：Developmental Dynamics  

Background: Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Results: Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Conclusion: Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

DOI： 10.1002/dvdy.352

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PubMed

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

During embryogenesis, different tissues develop coordinately, and this coordination is often in harmony with body growth. Recent studies allow us to understand how this harmonious regulation is achieved at the levels of inter-cellular, inter-tissue, and tissue-body relationships. Here, we present an overview of recently revealed mechanisms by which axial growth (tail growth) drives a variety of morphogenetic events, with a focus on the coordinated progression between Wolffian (nephric) duct elongation and somitogenesis. We also discuss how we can relate this coordination to the events occurring during limb bud outgrowth, since the limb buds and tail bud are appendage anlagen acquired during vertebrate evolution, both of which undergo massive elongation/outgrowth.

DOI： 10.1387/ijdb.170290yt

Language：English  

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog (Shh) is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 (Fgf8) is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis. Here we use cultured limb mesenchyme cells to assess the response of the target Hoxd genes to these two factors. We find that they act synergistically and that both factors are required to activate Hoxd13 in limb mesenchymal cells. However, the analysis of the enhancer landscapes flanking the HoxD cluster reveals that the bimodal regulatory switch observed in vivo is only partially achieved under these in vitro conditions, suggesting an additional requirement for other factors.

DOI： 10.1073/pnas.1620767114

Language：English   Publishing type：Research paper (bulletin of university, research institution)  

In most organs of the body, epithelial tissues are supported by their own basement membrane and underlying stroma, the latter being regarded as a complex of amorphous cells, extracellular matrices, and soluble factors. We demonstrate here that an epithelial tube can serve as a component of stroma that supports the formation of epithelial cell sheet derived from a different origin. During development of the mesonephros in chicken embryos, the intermediate mesoderm (IMM), which contains the Wolffian duct (WD) and its associated tubules, is overlain by a sheet of epithelial cells derived from lateral plate (coelomic) mesoderm. We describe that in normal embryos, epitheliogenesis of IMM tubes and the adjacent coelomic cell sheet proceed in a coordinated manner. When the WD was surgically ablated, the overlying coelomic epithelium exhibited aberrant morphology accompanied by a punctated basement membrane. Furthermore, the WD-ablated coelomic epithelium became susceptible to latent external stress; electroporation of Rac1 resulted in epithelial-to-mesenchymal transitions (EMTs) within the coelomic epithelium. The distorted coelomic epithelium was rescued by implanting fibronectin-producing cells in place of the WD, suggesting that fibronectin provided by WD has an important role acting interepithelially. This notion was corroborated further by directly visualizing a translocation of EGFP-tagged fibronectin from fibronectin-producing to -receiving epithelia in vivo. Our findings provide a novel insight into interepithelial signaling that also might occur in adult tissues to protect against EMT and suggest a possible new target for anticancer therapeutic strategy.

DOI： 10.1073/pnas.1316728111

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：奈良   Country：Japan  

Event date： 2020.6

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Venue：新潟   Country：Japan  

Event date： 2020.6

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Venue：横浜   Country：Japan  

Event date： 2020.6

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Venue：神戸   Country：Japan  

Event date： 2020.6

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Venue：京都   Country：Japan  

Event date： 2020.6

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Venue：アルバカーキ   Country：United States  

Event date： 2020.6

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Venue：奈良   Country：Japan  

Event date： 2020.6

Language：English  

Venue：宜野湾   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：松江   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：キール   Country：Germany  

Event date： 2020.6

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Venue：ミネアポリス   Country：United States  

Event date： 2020.6

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Venue：ポートランド（オレゴン）   Country：United States  

Event date： 2020.6

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Venue：横浜   Country：Japan  

Event date： 2020.6

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Venue：大阪   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：バルセロナ   Country：Spain  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：奈良   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Oral presentation (general)  

Venue：名古屋   Country：Japan  

Language：English  

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Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English  

Language：English   Presentation type：Oral presentation (general)  

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