| 1. |
Daisuke Saito, Ryosuke Tadokoro, Arata Nagasaka, Daisuke Yoshino, Takayuki Teramoto, Kanta Mizumoto, Kenichi Funamoto, Hinako Kidokoro, Takaki Miyata, Koji Tamura, Yoshiko Takahashi, Stiffness of primordial germ cells is required for their extravasation in avian embryos, iScience, 10.1016/j.isci.2022.105629, 105629-105629, iScience 20, 105629, 2022.11. |
| 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. |
Hidetaka Murai, Minami Shibuya, Ryohei Kishita, Chihiro Sunase, Koji Tamura, Daisuke Saito, Envelopment by endothelial cells initiates translocation of avian primordial germ cell into vascular tissue., Developmental dynamics : an official publication of the American Association of Anatomists, 10.1002/dvdy.332, 2021.03, BACKGROUND: In avian species, primordial germ cells (PGCs) migrate to the gonadal primordium through the vascular system. Because this mode of migration is reminiscent of cancer metastasis, it would be useful to elucidate the mechanisms underlying PGC migration via the bloodstream. Here, we sought to determine when, where, and how PGCs enter the vascular network by double visualization of PGCs and endothelial cells (ECs) in tie1:H2B-eYFP transgenic quails. RESULTS: In the left and right lateral germinal crescent regions corresponding to the anterior-most area vasculosa, more than 60% of PGCs were enveloped by differentiating ECs forming blood islands prior to vascular network formation. Cell morphology analysis suggested that the PGC-EC interaction was instructed by differentiating ECs. At a later developmental stage, ECs anastomosed to form a vascular network with a lumen that retained PGCs within it. As a consequence, many PGCs localized within the luminal space of the mature vascular network at later stages. CONCLUSIONS: Our findings demonstrate that the major type of avian PGC translocation into vascular tissue is not a typical intravasation, as performed by types of metastatic cancer cells, but rather a passive translocation (envelopment) mediated by differentiating ECs during early vasculogenesis.. |
| 4. |
Satoshi Yamanaka, Hidetaka Murai, Daisuke Saito, Gembu Abe, Etsuko Tokunaga, Takahiro Iwasaki, Hirotaka Takahashi, Hiroyuki Takeda, Takayuki Suzuki, Norio Shibata, Koji Tamura, Tatsuya Sawasaki, Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF., The EMBO journal, 10.15252/embj.2020105375, 40, 4, e105375, e2020105375, 2021.02, Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.. |
| 5. |
Morphogenetic mechanism of the acquisition of the dinosaur-type acetabulum.. |
| 6. |
Yoshino T, Saito D, Epithelial-to-mesenchymal transition-based morphogenesis of dorsal mesentery and gonad., Seminars in cell & developmental biology, 10.1016/j.semcdb.2018.09.002, 92, 105-112, 2018.09. |
| 7. |
Daisuke Saito, Koji Tamura, Yoshiko Takahashi, Early segregation of the adrenal cortex and gonad in chicken embryos, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/dgd.12389, 59, 7, 593-602, 2017.09, The adrenal gland is an endocrine organ that plays essential roles in stress responses. This organ consists of two types of tissues, adrenomedulla and adrenocortex, deriving from different embryonic origins. Whereas it is well accepted that the adrenomedulla derives from neural crest cells, the origin of the adrenocortex remains elusive. In addition, the adrenocortex and gonads, two major steroid hormone-producing tissues, have been thought to share the same origin, although the experimental evidence is lacking. In this study, to identify the origin of adrenocortex and to compare it to that of gonads, we scrutinized the medial portion of the coelomic epithelium (CE) after the lateral plate mesoderm has split into two CE components with a concomitant opening of the coelomic cavity in between them. We found that early medial CE consists of a two-cell layer-thick band of epithelial-like cells, the outer and inner CEs. The outer CE faces the coelomic cavity, whereas the inner CE is juxtaposed to nascent blood vessels. Combining direct cell labeling with early molecular markers, we found that outer CE was the origin of the gonad but not the adrenocortex. The adrenocortex, instead, appears to derive from inner CE. Thus, the adrenocortical and gonadal progenitors are already segregated from each other when the coelomic cavity has opened. This study provides a new basis for understanding how the adrenal gland forms and how steroid hormone-producing tissues arise during development.. |
| 8. |
Haruka Matsubara, Daisuke Saito, Gembu Abe, Hitoshi Yokoyama, Takayuki Suzuki, Koji Tamura, Upstream regulation for initiation of restricted Shh expression in the chick limb bud, DEVELOPMENTAL DYNAMICS, 10.1002/dvdy.24493, 246, 5, 417-430, 2017.05, Background: The organizing center, which serves as a morphogen source, has crucial functions in morphogenesis in animal development. The center is necessarily located in a certain restricted area in the morphogenetic field, and there are several ways in which an organizing center can be restricted. The organizing center for limb morphogenesis, the ZPA (zone of polarizing activity), specifically expresses the Shh gene and is restricted to the posterior region of the developing limb bud.Results: The pre-pattern along the limb anteroposterior axis, provided by anterior Gli3 expression and posterior Hand2 expression, seems insufficient for the initiation of Shh expression restricted to a narrow, small spot in the posterior limb field. Comparison of the spatiotemporal patterns of gene expression between Shh and some candidate genes (Fgf8, Hoxd10, Hoxd11, Tbx2, and Alx4) upstream of Shh expression suggested that a combination of these genes' expression provides the restricted initiation of Shh expression.Conclusions: Taken together with results of functional assays, we propose a model in which positive and negative transcriptional regulatory networks accumulate their functions in the intersection area of their expression regions to provide a restricted spot for the ZPA, the source of morphogen, Shh. Developmental Dynamics 246:417-430, 2017. (c) 2017 Wiley Periodicals, Inc.. |
| 9. |
Ryohei Seki, Cai Li, Qi Fang, Shinichi Hayashi, Shiro Egawa, Jiang Hu, Luohao Xu, Hailin Pan, Mao Kondo, Tomohiko Sato, Haruka Matsubara, Namiko Kamiyama, Keiichi Kitajima, Daisuke Saito, Yang Liu, M. Thomas P. Gilbert, Qi Zhou, Xing Xu, Toshihiko Shiroishi, Naoki Irie, Koji Tamura, Guojie Zhang, Functional roles of Aves class-specific cis-regulatory elements on macroevolution of bird-specific features, NATURE COMMUNICATIONS, 10.1038/ncomms14229, 8, 14229, 2017.02, Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (> 99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.. |
| 10. |
Yoshino, T, Murai, H, Saito, D, Hedgehog–BMP signalling establishes dorsoventral patterning in lateral plate mesoderm to trigger gonadogenesis in chicken embryos., Nat Commun, 10.1038/ncomms12561, 7, 12561, 12561, 2016.08. |
| 11. |
Seki R, Kitajima K, Matsubara H, Suzuki T, Saito D, Yokoyama H, Tamura K, AP-2β is a transcriptional regulator for determination of digit length in tetrapods., Developmental biology, 10.1016/j.ydbio.2015.08.006, 407, 1, 75-89, 2015.11. |
| 12. |
Daisuke Saito, Yoshiko Takahashi, Sympatho-adrenal morphogenesis regulated by the dorsal aorta, MECHANISMS OF DEVELOPMENT, 10.1016/j.mod.2015.07.011, 138, 2-7, Review, 2015.11, The autonomic nervous system, composed of sympathetic-and para-sympathetic neurons, plays essential roles in a variety of physiological functions including homeostasis and responses to external stimuli. We here present an overview of recent findings concerning how the sympathetic nervous system is formed during the early development, paying particular attention to the morphogenesis of those tissues derived from migrating neural crest cells. Neural crest cells, originally multipotent, are progressively specified to sympathetic ganglia neurons and adrenomedullary cells during their migration through the body. Importantly, the dorsal aorta, the first-forming blood vessel, acts as a signaling center for their migration and differentiation. BMP signals emanating from the dorsal aorta are essential for establishing environmental cues that directly act on the migrating cells. The mechanisms underlying these early neuro-vascular interactions provide insights into understanding diseases caused by malfunctions and malformations of the autonomic nervous system. (C) 2015 Elsevier Ireland Ltd. All rights reserved.. |
| 13. |
Teruaki Takahashi, Yuta Takase, Takashi Yoshino, Daisuke Saito, Ryosuke Tadokoro, Yoshiko Takahashi, Angiogenesis in the Developing Spinal Cord: Blood Vessel Exclusion from Neural Progenitor Region Is Mediated by VEGF and Its Antagonists, PLOS ONE, 10.1371/journal.pone.0116119, 10, 1, e0116119, e0116119, 2015.01, Blood vessels in the central nervous systemsupply a considerable amount of oxygen via intricate vascular networks. We studied how the initial vasculature of the spinal cord is formed in avian (chicken and quail) embryos. Vascular formation in the spinal cord starts by the ingression of intra-neural vascular plexus (INVP) from the peri-neural vascular plexus (PNVP) that envelops the neural tube. At the ventral region of the PNVP, the INVP grows dorsally in the neural tube, and we observed that these vessels followed the defined path at the interface between the medially positioned and undifferentiated neural progenitor zone and the laterally positioned differentiated zone. When the interface between these two zones was experimentally displaced, INVP faithfully followed a newly formed interface, suggesting that the growth path of the INVP is determined by surrounding neural cells. The progenitor zone expressed mRNA of vascular endothelial growth factor-A whereas its receptor VEGFR2 and FLT-1 (VEGFR1), a decoy for VEGF, were expressed in INVP. By manipulating the neural tube with either VEGF or the soluble form of FLT-1, we found that INVP grew in a VEGF-dependent manner, where VEGF signals appear to be fine-tuned by counteractions with anti-angiogenic activities including FLT-1 and possibly semaphorins. These results suggest that the stereotypic patterning of early INVP is achieved by interactions between these vessels and their surrounding neural cells, where VEGF and its antagonists play important roles.. |
| 14. |
Takashi Yoshino, Daisuke Saito, Yuji Atsuta, Chihiro Uchiyama, Shinya Ueda, Kiyotoshi Sekiguchi, Yoshiko Takahashi, 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.. |
| 15. |
Yuji Atsuta, Ryosuke Tadokoro, Daisuke Saito, Yoshiko Takahashi, Transgenesis of the Wolffian duct visualizes dynamic behavior of cells undergoing tubulogenesis in vivo., Development, growth & differentiation, 10.1111/dgd.12047, 55, 4, 579-90, 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.. |
| 16. |
Daisuke Saito, Yuta Takase, Hidetaka Murai, Yoshiko Takahashi, The Dorsal Aorta Initiates a Molecular Cascade That Instructs Sympatho-Adrenal Specification, SCIENCE, 10.1126/science.1222369, 336, 6088, 1578-1581, 2012.06, The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF 1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.. |
| 17. |
Yasuhiro Yokota, Daisuke Saito, Ryosuke Tadokoro, Yoshiko Takahashi, Genomically integrated transgenes are stably and conditionally expressed in neural crest cell-specific lineages, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2011.02.001, 353, 2, 382-395, 2011.05, Neural crest cells (NCCs) are a transient embryonic structure that gives rise to a variety of cells including peripheral nervous system. melanocytes, and Schwann cells. To understand the molecular mechanisms underlying NCC development, a gene manipulation of NCCs by in ovo electroporation technique is a powerful tool, particularly in chicken embryos, the model animal that has long been used for the NCC research. However, since expression of introduced genes by the conventional electroporation method is transient, the mechanisms of late development of NCCs remain unexplored. We here report novel methods by which late-developing NCCs are successfully manipulated with electroporated genes. Introduced genes can be stably and/or conditionally expressed in a NCC-specific manner by combining 4 different techniques: Tol2 transposon-mediated genomic integration (Sato et al., 2007), a NCC-specific enhancer of the Sox10 gene (identified in this study), Cre/loxP system, and tet-on inducible expression (Watanabe et al., 2007). This is the first demonstration that late-developing NCCs in chickens are gene-manipulated specifically and conditionally. These methods have further allowed us to obtain ex vivo live-images of individual Schwann cells that are associated in axon bundles in peripheral tissues. Cellular activity and morphology dynamically change as development proceeds. This study has opened a new way to understand at the molecular and cellular levels how late NCCs develop in association with other tissues during embryogenesis. (C) 2011 Elsevier Inc. All rights reserved.. |
| 18. |
Takashi Yoshino, Daisuke Saito, Yoshiko Takahashi, Epithelial integrity requires a signal from underlying stroma: The nephric coelomic epithelium as a novel experimental model, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2008.05.449, 319, 2, 605-605, 2008.07. |
| 19. |
Takashi Yoshino, Daisuke Saito, Yoshiko Takahashi, Coordinated formation between fibronectin-producing and fibronectin-receiving epithelia, DIFFERENTIATION, 10.1016/j.diff.2010.09.098, 80, S48-S48, 2010.11. |
| 20. |
Daisuke Saito, Emi Ohata, Hidetaka Murai, Yuta Takase, Yoshiko Takahashi, BMP-switching regulates lineage specification and migration of neural crest cells, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2010.05.238, 344, 1, 473-473, 2010.08. |
| 21. |
Takashi Yoshino, Daisuke Saito, Ryosuke Tadokoro, Yoshiko Takahashi, In vivo gene manipulations of epithelial cell sheets: A novel model to study epithelial-to-mesenchymal transition, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/j.1440-169X.2011.01252.x, 53, 3, 378-388, 2011.04, Embryonic cells are classified into two types of cells by their morphology, epithelial and mesenchymal cells. During dynamic morphogenesis in development, epithelial cells often switch to mesenchymal by the process known as epithelial-to-mesenchymal transition (EMT). EMT is a central issue in cancer metastasis where epithelial-derived tumor cells are converted to mesenchymal with high mobility. Although many molecules have been identified to be involved in the EMT mostly by in vitro studies, in vivo model systems have been limited. We here established a novel model with which EMT can be analyzed directly in the living body. By an electroporation technique, we targeted a portion of the lateral plate mesoderm that forms epithelial cell sheets delineating the kidney region, called nephric coelomic epithelium (Neph-CE). Enhanced green fluorescent protein-electroporated Neph-CE retained the epithelial integrity without invading into the underling stroma (mesonephros). The Neph-CE transgenesis further allowed us to explore EMT inducers in vivo, and to find that Ras-Raf and RhoA signals were potent inducers. Live-imaging confocal microscopy revealed that during EMT processes cells started extending cellular protrusions toward the stroma, followed by translocation of their cell bodies. Furthermore, we established a long-term tracing of EMT-induced cells, which were dynamically relocated within the kidney stroma. The Neph-CE-transgenesis will open a way to study cellular and molecular mechanisms underlying EMT directly in actual body.. |
| 22. |
Emi Ohata, Ryosuke Tadokoro, Yuki Sato, Daisuke Saito, Yoshiko Takahashi, Notch signal is sufficient to direct an endothelial conversion from non-endothelial somitic cells conveyed to the aortic region by CXCR4, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2009.08.010, 335, 1, 33-42, 2009.11, 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 naive mesenchymal cells to differentiate into endothelial cells once the cells are conveyed to the aortic region. (C) 2009 Elsevier Inc. All rights reserved.. |
| 23. |
Tadayoshi Watanabe, Yuki Sato, Daisuke Saito, Ryosuke Tadokoro, Yoshiko Takahashi, EphrinB2 coordinates the formation of a morphological boundary and cell epithelialization during somite segmentation, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.0902859106, 106, 18, 7467-7472, 2009.05, 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, up-regulates 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.. |
| 24. |
Yuki Sato, Tadayoshi Watanabe, Daisuke Saito, Teruaki Takahashi, Shosei Yoshida, Jun Kohyama, Emi Ohata, Hideyuki Okano, Yoshiko Takahashi, Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos, DEVELOPMENTAL CELL, 10.1016/j.devcel.2008.03.024, 14, 6, 890-901, 2008.06, We studied, using avian embryos, mechanisms underlying the three-dimensional assembly of the dorsal aorta, the first-forming embryonic vessel in amniotes. This vessel originates from two distinct cell populations, the splanchnic and somitic mesoderms. We have unveiled a role for Notch signaling in the somitic contribution. Upon activation of Notch signaling, a subpopulation of cells in the posterior half of individual somites migrates ventrally toward the primary dorsal aorta of splanchnic origin. After reaching the primary aorta, these somitic cells differentiate into the definitive aortic endothelial cells. This Notch-induced ventral migration is mediated by EphrinB2 and by an attractant action of the primary aorta. Furthermore, long-term chasing of cells by transposon-mediated gene transfer reveals that the segmentally provided endothelial cells of somitic origin in the dorsal aorta ultimately populate the entire region of the vessel. We demonstrate the molecular and cellular mechanisms underlying the formation of embryonic blood vessels from mesenchymal cells.. |
| 25. |
Tadayoshi Watanabe, Daisuke Saito, Koji Tanabe, Rinako Suetsugu, Yukiko Nakaya, Shinichi Nakagawa, Yoshiko Takahashi, Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2007.01.042, 305, 2, 625-636, 2007.05, The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off', works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. (c) 2007 Elsevier Inc. All rights reserved.. |
| 26. |
D Saito, S Yonei-Tamura, Y Takahashi, K Tamura, Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2006.01.002, 292, 1, 79-89, 2006.04, Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the pat-axial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and downregulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and downregulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation. (c) 2006 Elsevier Inc. All rights reserved.. |
| 27. |
K Tamura, T Amano, T Satoh, D Saito, S Yonei-Tamura, H Yajima, Expression of rigf, a member of avian VEGF family, correlates with vascular patterning in the developing chick limb bud, MECHANISMS OF DEVELOPMENT, 10.1016/S0925-4773(02)00411-2, 120, 2, 199-209, 2003.02, In a differential display screening for genes regulated by retinoic acid in the developing chick limb bud, we have isolated a novel gene, termed rigf, retinoic-acid induced growth factor, that encodes a protein belonging to the vascular endothelial growth factor (VEGF) family. Rigf transcripts were found in the posterior region of the limb bud in a region-specific manner as well as in other embryonic tissues and regions, including the notochord, head and trunk mesenchyme, retinal pigment epithelium, and branchial arches. Several manipulations revealed that retinoic acid and sonic hedgehog signaling pathways regulate rigf expression in the limb bud. VEGF family members, which promote the migration, differentiation and proliferation of endothelial cells in both blood and lymphatic vessels, are important factors for the formation of blood and lymphatic vasculatures during development. We demonstrated that the anterior border of the rigf expression domain in the limb bud corresponds with the position of the primary central artery (the subclavian artery in the forelimb), which is a main artery for supplying blood to the limb. These observations taken together with results from some experimental manipulations suggest that the limb tissue attracts blood vessels into the limb bud and that rigf is involved in the pattern formation of blood vessels in the limb. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.. |
| 28. |
D Saito, S Yonei-Tamura, K Kano, H Ide, K Tamura, Specification and determination of limb identity: evidence for inhibitory regulation of Tbx gene expression, DEVELOPMENT, 129, 1, 211-220, 2002.01, Limb-type-specific expression of Tbx5/Tbx4 plays a key role in drawing distinction between a forelimb and a hindlimb. Here, we show insights into specification and determination during commitment of limb-type identity, in particular that median tissues regulate Tbx expressions. By using the RTPCR technique on chick embryos, the onset of specific Tbx5/Tbx4 expression in the wing/leg region was estimated to be stage 13. Specification of the limb-type identity is thought to occur before stage 9, since all explants from stage 9 through 14 expressed the intrinsic Tbx gene autonomously in a simple culture medium. The results of transplantation experiments revealed that axial structures medial to the lateral plate mesoderm at the level of the wing region are capable of transforming leg identity to wing identity, suggesting that a factor(s) from the median tissues is involved in the limb-type determination. Nevertheless, the transplanted wing region was not converted to leg identity. The results of the transplantation experiments also suggested that wing-type identity is determined much earlier than is leg-type identity. Finally, we also found that inhibitory effects of median tissues mediate the specific expression of Tbx5/Tbx4 in the presumptive wing/leg region. We propose a model for limb-type identification in which inhibitory regulation is involved in restricting one Tbx gene expression by masking the other Tbx expression there.. |
| 29. |
K Tamura, R Kuraishi, D Saito, H Masaki, H Ide, S Yonei-Tamura, Evolutionary aspects of positioning and identification of vertebrate limbs, JOURNAL OF ANATOMY, 10.1017/S0021878201008081, 199, 195-204, 2001.07, Emerging developmental studies contribute to our understanding of vertebrate evolution because changes in the developmental process and the genes responsible for such changes provide a unique way for evaluating the evolution of morphology. Endoskeletal limbs, the locomotor organs that are unique to vertebrates, are a popular model system in the fields of palaeontology and phylogeny because their structure is highly visible and their bony pattern is easily preserved in the fossil records. Similarly, limb development has long served as an excellent model system for studying vertebrate pattern formation. In this review, the evolution of vertebrate limb development is examined in the light of the latest knowledge, viewpoints and hypotheses.. |
