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To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.019 Byline: Atsuko Yamada (a), Mark Q. Martindale (b), Akimasa Fukui (c), Shin Tochinai (a) Keywords: Brachyury; Ctenophore; Xenopus; Morphogenetic movements; Convergent extension Abstract: Brachyury, a member of the T-box transcription family identified in a diverse array of metazoans, was initially recognized for its function in mesoderm formation and notochord differentiation in vertebrates; however, its ancestral role has been suggested to be in control of morphogenetic movements. Here, we show that morpholino oligonucleotide knockdown of Brachyury (MlBra) in embryos of a ctenophore, one of the most ancient groups of animals, prevents the invagination of MlBra expressing stomodeal cells and is rescued with corresponding RNA injections. Injection of RNA encoding a dominant-interfering construct of MlBra causes identical phenotypes to that of RNA encoding a dominant-interfering form of Xenopus Brachyury (Xbra) in Xenopus embryos. Both injected embryos down-regulate Xbra downstream genes, Xbra itself and Xwnt11 but not axial mesodermal markers, resulting in failure to complete gastrulation due to loss of convergent extension movements. Moreover, animal cap assay reveals that MlBra induces Xwnt11 like Xbra. Overall results using Xenopus embryos show that these two genes are functionally interchangeable. These functional experiments demonstrate for the first time in a basal metazoan that the primitive role of Brachyury is to regulate morphogenetic movements, rather than to specify endomesodermal fates, and the role is conserved between non-bilaterian metazoans and vertebrates. Author Affiliation: (a) Department of Natural History Sciences, Faculty of Science, Hokkaido University, N10 W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan (b) Kewalo Marine Laboratory, Pacific Bioscience Research Center, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813, USA (c) Department of Biological Sciences, Graduate School of Science, Hokkaido University, N10 W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan Article History: Received 26 June 2009; Revised 31 October 2009; Accepted 14 December 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.03.042 Byline: Yuzuru Ito (a), Satsuki Seno (b), Hiroaki Nakamura (c), Akimasa Fukui (b), Makoto Asashima (a)(b)(d) Keywords: HAPLN3; Heart development; Hyaluronan; has2; Versican; Xenopus Abstract: Hyaluronan matrix plays an important role during vertebrate cardiogenesis. Transcripts for the hyaluronan synthase Has2 gene are expressed in heart anlage, and disruption of either Has2 or versican, a hyaluronan matrix component, abrogates normal cardiac morphogenesis. However, the mechanisms by which hyaluronan matrix contributes to early heart development are largely unknown. Here we show that Xenopus hyaluronan and proteoglycan-binding link protein 3 (XHAPLN3) helps to maintain hyaluronan matrix around the cardiac anlage, and thereby contribute to cardiogenesis. XHAPLN3 mRNA transcript localization overlapped with the mRNA expression of both Xhas2 and Xversican at the heart anlage of early tailbud (stage 23) embryos. Furthermore, knockdown of XHAPLN3 or Xhas2 with morpholino antisense oligos caused a heart deficiency in developing tadpoles. Our results show when and how components of the hyaluronan matrix function in cardiogenesis, improving our understanding of how extracellular matrix participates in embryogenesis. Author Affiliation: (a) ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan (b) Department of Life Science (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan (c) Department of Oral Histology, Matsumoto Dental University, 1780 Gobara, Hirooka, Shiojiri, Nagano 399-0781, Japan (d) Organ Development Research Laboratory, National Institute of Advanced Industrial Sciences and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan Article History: Received 10 October 2007; Revised 22 February 2008; Accepted 31 March 2008

Byline: Motohiro Homma (a), Masafumi Inui (a), Akimasa Fukui (a), Tatsuo Michiue (a)(b), Koji Okabayashi (c), Makoto Asashima (a)(b)(c) Keywords: Xenopus laevis; Convergent extension; Activin-like signaling; BENI; Xbra; Nuclear localization signal (NLS) Abstract: Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-[beta] family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression. Author Affiliation: (a) Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan (b) AIST (Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan (c) ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan Article History: Received 29 November 2005; Revised 31 October 2006; Accepted 7 November 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.028 Byline: Masafumi Inui (a), Akimasa Fukui (b), Yuzuru Ito (c), Makoto Asashima (b)(c)(d) Keywords: Apelin; Xmsr; GPCR; APJ; Heart; Endothelium; Blood; XlFli Abstract: The cardiovascular development is the elaborate process, and despite the extensive studies, the mechanisms underlying endothelial, hematopoietic, and cardiac developments, as well as the interrelation between these processes, are not fully understood. In this study, we demonstrated that Xenopus apelin and Xmsr play pivotal roles in cardiovascular development. Apelin is a recently identified ligand for an orphan G-protein-coupled receptor APJ and is involved in fluid homeostasis in mammals. Xenopus preproapelin (Xpreproapelin) was isolated and its mRNA localized to the region around the presumptive blood vessels, which are overlapping or adjacent to those expressing Xmsr, the Xenopus homologue of APJ. Overexpression of Xpreproapelin disorganized the expression of the endothelial precursor cell marker XlFli and the hematopoietic precursor cell marker SCL at the neurula, whereas embryos injected with morpholino antisense oligonucleotides for Xapelin and Xmsr displayed attenuated expression of Tie2, [alpha]-globin, XPOX2, and cTnI, markers of endothelium, erythrocytes, myeloid cells, and cardiomyocytes, respectively. XlFli morpholino had similar effects to Xapelin and Xmsr morpholinos on cardiac differentiation, suggesting an unexpected potential relationship between the endothelium and cardiac differentiation. Forced expression of constitutive active G[alpha]i rescued the phenotypes of Xmsr morpholino-injected embryos, indicating that the i/o type of G protein [alpha] subunit acts downstream of Xmsr. Author Affiliation: (a) Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan (b) Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan (c) International Cooperative Research Project (ICORP), Japan Science and Technology Agency (JST), Tokyo, Japan (d) National Institute of Advanced Science and Technology (AIST), Organ Development Research Laboratory, Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan Article History: Received 9 January 2006; Revised 11 June 2006; Accepted 15 June 2006

Abstract Background Persistent infection of human papillomavirus (HPV) types 16 and 18 causes cervical cancer. To better understand immune responses to the prophylactic vaccine, HPV 16/18 L1 virus-like particles (HPV-VLPs), we investigated B cell epitopes of HPV16 L1-derived peptides. Methods Sera from mice immunized with HPV-16/18 L1 VLPs were analyzed for their IgG titers against 10 different HPV16 L1-derived peptides (20-mer) that contain human leukocyte antigen (HLA)-class I A-2, A-24 and class II DR. Results One 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2d) and C57BL/6 (H-2b) mice. Mapping analysis showed that the 10-amino-acid sequence at positions 304to 313 was an immunogenic portion. It is of note that the binding capability of this 10-mer peptide to the HLA-A2 and HLA-A24 molecules was confirmed by the HLA class I stabilization assay. In addition, one unique 20-mer was determined as a B cell epitope in each strain. Conclusions These results might provide new information for better understanding of immune responses to HPV 16 L1.

Early Xenopus embryos contain follistatin, an activin-binding protein, and three isoforms of activin, B, AB and A. Induction of mesoderm during early Xenopus embryogenesis is influenced by activin present in the eggs. The role of activin in signaling developmental variations can be regulated by follistatin. Cellular content of the isoforms and follistatin differ from each other.