| 1. |
Daniel Aldea, Blerina Kokalari, Yuji Atsuta, Heather L. Dingwall, Ying Zheng, Arben Nace, George Cotsarelis, Yana G. Kamberov, Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development, PLoS Genetics, 10.1371/journal.pgen.1010614, 2023.02. |
| 2. |
Yuji Atsuta, Katsuya Suzuki, Hiroko Iikawa, Haruna Yaguchi, Daisuke Saito, Prime editing in chicken fibroblasts and primordial germ cells, Development, Growth & Differentiation, 10.1111/dgd.12823, 2022.11. |
| 3. |
Alan R Rodrigues, Nayuta Yakushiji-Kaminatsui, Yuji Atsuta, Guillaume Andrey, Patrick Schorderet, Denis Duboule, Clifford J Tabin, Integration of Shh and Fgf signaling in controlling Hox gene expression in cultured limb cells., Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1620767114, 114, 12, 3139-3144, 2017.03, 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.. |
| 4. |
Atsuta, Yuji; Tomizawa, Reiko R.; Levin, Michael; Tabin, Clifford J., L-type voltage-gated Ca2+ channel Ca(V)1.2 regulates chondrogenesis during limb development, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1908981116, 116, 43, 21592-21601, 2019.10, 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.. |
| 5. |
Takahashi, Yoshiko; Kudo, Ryo; Tadokoro, Ryosuke; Atsuta, Yuji, Coordination between body growth and tissue growth: Wolffian duct elongation and somitogenesis proceed in harmony with axial growth, INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY, 10.1387/ijdb.170290yt, 62, 1-3, 79-84, 2018.05, 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.. |
| 6. |
Atsuta, Yuji; Takahashi, Yoshiko, Early formation of the Mullerian duct is regulated by sequential actions of BMP/Pax2 and FGF/Lim1 signaling, DEVELOPMENT, 10.1242/dev.137067, 143, 19, 3549-3559, 2016.10, 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.. |
| 7. |
Atsuta, Yuji; Takahashi, Yoshiko, FGF8 coordinates tissue elongation and cell epithelialization during early kidney tubulogenesis, DEVELOPMENT, 10.1242/dev.122408, 142, 13, 2329, 2015.07, 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.. |
| 8. |
Yoshino, Takashi; Saito, Daisuke; Atsuta, Yuji; Uchiyama, Chihiro; Ueda, Shinya; Sekiguchi, Kiyotoshi; Takahashi, Yoshiko, Interepithelial signaling with nephric duct is required for the formation of overlying coelomic epithelial cell sheet, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1316728111, 111, 18, 6660-6665, 2014.05, 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.. |
| 9. |
Atsuta, Yuji; Tadokoro, Ryosuke; Saito, Daisuke; Takahashi, Yoshiko, Transgenesis of the Wolffian duct visualizes dynamic behavior of cells undergoing tubulogenesis in vivo, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/dgd.12047, 55, 4, 579-590, 2013.05, 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.. |
| 10. |
Daniel Aldea*, Yuji Atsuta*, Blerina Kokalari, Stephen F. Schaffner, Rexxi D. Prasasya,
Adam Aharoni, Heather L. Dingwall, Bailey Warder, Yana G. Kamberov (*equal contribution), Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.2021722118, 118, 16, e2021722118, 2021; Vol. 118 (No. 16): e2021722118, 2021.04, Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans’ high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification ofmore eccrine glands. Here, we show that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, we identified a human EN1 skin enhancer, hECE18. We showed that multiple epistatically interacting derived substitutions in the human ECE18 enhancer increased its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. Our study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.. |
| 11. |
Reiko R. Tomizawa, Clifford J. Tabin, Yuji Atsuta (corresponding author), In ovo electroporation of chicken limb bud ectoderm, Developmental Dynamics, in press, 2021.05. |
