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Direct contributions of Otx2 as a positional tag to global gene regulation in the head organizer in Xenopus embryos Yuuri Yasuoka1, Yutaka Suzuki2, Shuji Takahashi3, Norihiro Sudou1, Yoshikazu Haramoto4, Makoto Asashima4, Sumio Sugano2, and Masanori Taira1 1Dept of Biol Sci, Grad Sch of Sci; 2Dept of Med Genome Sci, Grad Sch of Front Sci; 3Div of Life Sci, College of Arts and Sci, Univ. of Tokyo; 4SCRC, Nat Inst of Adv Industrial Sci and Tech, Japan Bilaterians use the homeodomain protein Otx to form the head, but how its positional information is utilized remains uncertain. In vertebrates, both Otx2 and the LIM homeodomain protein Lim1/Lhx1 are required for head formation, but the regulatory principles underlying Lim1 and Otx2 functions in the head organizer remain unsolved. In this talk, I present our recent data using ChIP-seq analysis, showing that Otx2, Lim1, the coactivator p300, and the corepressor TLE/Groucho colocalize on cis-regulatory modules (CRMs) of thousands of genes including most ‘head-organizer’ genes and even most ‘non-head-organizer’ genes in the Xenopus tropicalis gastrula. Comprehensive analysis of CRMs with RNA-seq data revealed that Lim1/Otx2-bound CRMs co-localizing with TLE rather than p300 are strongly associated with region/tissue-specific genes. Together with reporter analyses, our data suggest that Otx2 functions as a ‘positional tag’ for a number of genes, in which Otx2 not only activates headorganizer genes in association with Lim1 but also represses non-head-organizer genes with transcriptional repressors such as Goosecoid. Thus, it is likely that each of thousands of genes interprets Otx2 as a positional tag to determine its expression in the head organizer. Our ‘positional tag hypothesis’ provides the idea that positional information conducts ‘distributive regulation’ of many genes, resulting in regional identity by the summation of these individual responses, which is contrast to ‘top-down control’ as the top of regulatory hierarchy defining regional identity. Based on these data, we are also analyzing histone modifications, H3K4me1 and H3K27Ac, for enhancer markers in comparison with Otx2/Lim1/TLE-bound CRM types and expression profiles, which will be discussed in the end of my talk. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Epigenetic regulation of key developmental genes in medaka Hiroyuki Takeda Department of Biological Sciences, Graduate School of Science, University of Tokyo In ES cells, “key developmental genes” are marked by specific modification called ‘bivalent’, co-existence of active (H3K4me) and repressive (H3K27me) histone modifications. The bivalent state becomes univalent (active or repressive) upon differentiation. Although extensive researches have been conducted using ES cells, in vivo evidence of this bivalency is still limited. Furthermore, the relationship between histone and DNA methylation has not been well studied thus far. To clarify these issues, we performed the genome-wide DNA methylome analysis as well as ChIP-seq analysis of H3K4me and H3K27me using the undifferentiated blastula-stage embryos of medaka fish. We found that most of the genomic DNA is highly methylated but loci where H3K4 and/or H3K27 methylation occur are hypomethylated. Among them, ~200 loci possess long DNA hypomethylated domain (>5kb) with bivalent histone modifications. Interestingly, ~75 % of genes within such loci were found to be key developmental genes including transcriptional regulators such as homeobox and T-box genes. The same analysis using adult liver and muscle revealed that DNA methylation is up-regulated, except for H3K4me-rich promoter regions, in actively transcribed loci. The further analysis focused on the zic gene, one of these loci, demonstrated that DNA methylation proceeds after hatching, whereas histone modification is established during embryonic stages. We propose that key developmental genes are regulated in a step-wise manner throughout life, bivalent and DNA hypomethylated state in undifferentiated early embryos, histone-modification-dependent induction during development and finally the maintenance of transcription by DNA-methylation after birth. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) DNA oxidation towards totipotency in mammalian development Guoliang Xu Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai Mammalian development starts with the fertilization of sperm and eggs that carry distinctive epigenetic modifications that are adjusted through chromatin remodeling. The paternal genome in the zygote undergoes active DNA demethylation before the first mitotic cell division. The biological significance and mechanisms of this early epigenome remodeling have remained unclear. We find that, within mouse zygotes, DNA oxidation of occurs on the paternal genome, changing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC). In Tet3-deficient zygotes from conditional knockout mice, the oxidation of 5mC fails to occur. Thus, the loss of 5mC in the paternal genome in developing zygotes is caused by Tet3-mediated oxidation. Deficiency of Tet3 impedes demethylation at the paternal Oct4 and Nanog genes and delays their subsequent reactivation in early embryos. Heterozygous mutant embryos derived from occytes lacking the maternal Tet3 suffer increased developmental failures, with female mice depleted of Tet3 in the germ line displaying severely reduced fecundity. Importantly, oocytes lacking Tet3 show impaired reprogramming of injected somatic cell nuclei. We conclude that Tet3-mediated DNA oxidation is essential for epigenetic reprogramming in the early embryo following natural fertilization, and also for the reprogramming of somatic cell nuclei during animal cloning. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Dynamic regulation of 5mC during preimplantation embryonic development Yi Zhang Howard Hughes Medical Institute, and Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Epigenetic modifications play important roles in diverse biological processes that range from regulation of gene expression, embryonic development, stem cell reprogramming, and human diseases such as cancers. One of the epigenetic modifications is DNA methylation. Although enzymes responsible for DNA methylation have been well characterized, enzymes that responsible for active DNA demethylation in mammalian cells have remained elusive. Recent studies have demonstrated that a novel family of proteins Tet1-3 have the capacity to convert 5mC to 5hmC, 5fC, and 5caC raising the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation. In this talk, I will present our recent findings on the dynamics of Tet oxidation products in preimplantation embryos and their potential role in early embryonic development. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Role of small RNAs in shaping the epigenome of germ cells for genome defense and imprinting Hiroyuki Sasaki Division of Epigenomics, Medical Institute of Bioregulation, Kyushu University During mammalian germ cell development, retrotransposons and many unique sequences including imprint control regions are de novo DNA methylated. This predominantly occurs at the prospermatogonium stage in the fetal testis. The global de novo methylation is important for maintenance of genomic integrity and regulation of developmental genes including imprinted genes in the offspring. De novo DNA methyltransferases Dnmt3a and Dnmt3b, and a related protein Dnmt3L, are responsible for this process, but how specific sequences are selected for methylation is not fully understood. In fission yeast and plants, examples are known where small interfering RNAs serves as a guide to recruit epigenetic modifiers to specific targets. We were therefore interested in looking at the role of small RNAs in shaping the epigenome of germ cells. We found that the imprint control region of the mouse Rasgrf1 locus lost methylation in spermatogonia deficient for MitoPLD, a component of the Piwi-interacting RNA (piRNA) pathway. A retrotransposon sequence in a novel non-coding RNA spanning the imprint control region was targeted by piRNAs generated from a different locus. Furthermore, a tandem repeat region required for methylation of the Rasgrf1 imprint control region was found to be the promoter for this non-coding RNA. We therefore propose a model in which piRNAs and its target non-coding RNA play a critical role in DNA methylation and imprinting of Rasgrf1. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) A systems approach reveals the molecular network of musculoskeletal development and homeostasis Hiroshi Asahara Department of Systems BioMedicine, Tokyo Medical and Dental University, National Research Institute for Child Health and Development We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS(http:// embrys.jp), containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis (FIG.1). This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58mediated repression of muscle regulatory factor (MRF) inhibitors. We applied our systems approaches to other musculoskeletal tissues research including cartilage and tendon, and revealed novel molecular network regulating joint cartilage development and homeostasis via miRNA-140 (Genes Dev, 2010; Arthritis Rheum, 2009) and tendon development by Mkx (Proc Natl Acad Sci U S A, 2010). References: Yokoyama S, et al., Dev Cell, 2009. Miyaki S, et al., Genes Dev, 2010. Ito Y, et al., Proc Natl Acad Sci U S A, 2010. FIG.1 Whole-mount in situ hybridization database “EMBRYS” Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Genome-wide analysis of protein-DNA interactions Arttu Jolma1,2, Jian Yan1, Thomas Whitington1, Martin Enge1, Kazuhiro Nitta1,3, Teemu Kivioja1,2, Mikko Taipale1, Juan M. Vaquerizas4, Nicholas M. Luscombe4, Minna Taipale1,2, Esko Ukkonen2, Yutaka Satou5, Patrick Lemaire3,6 and Jussi Taipale1,2 1Karolinska Institutet, Department of Biosciences and Nutrition; 2University of Helsinki, Finland;3IBDML, Marseilles, France; 4EMBL - European Bioinformatics Institute, Cambridge, UK; 5Department of Zoology, University of Kyoto; 6CRBM, MONTPELLIER, France Understanding the information encoded in the human genome requires two genetic codes, the first code specifies how mRNA sequence is converted to protein sequence, and the second code determines where and when the mRNAs are expressed. Although the proteins that read the second, regulatory code – transcription factors (TFs) – have been largely identified, the code is poorly understood as it is not known which sequences TFs can bind in the genome. To understand the regulatory code, we have analyzed the occupancy of the majority of all expressed TFs in human colorectal cancer cells, and analyzed the sequence-specific binding of all human TFs using high-throughput SELEX. Comparison of the human binding profiles with those of house mouse and an early chordate, Ciona intestinalis, revealed that the monomer binding specificity of TFs evolves very slowly, and has been almost completely fixed in the entire chordate lineage, despite complete lack of regulatory element conservation between Ciona and Human. However, factors that bind identical sequences as monomers form dimers with different spacing and orientation preferences, that and these preferences appear to evolve much faster than monomer binding preferences, indicating that changes in spacing and orientation preferences of TFs is a potential source of evolutionary novelty. A binding model that is required to understand binding of TFs to the genome, which incorporates information about protein-protein interactions induced by DNA, and inheritance of epigenetic states across cell division will be discussed. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) A genome-wide approach for understanding of the development of the Ciona embryos Yutaka Satou Department of Zoology, Graduate School of Science, Kyoto University After we determined the genome sequence of Ciona intestinalis, we have taken a genome-based approach to understand mechanisms behind the embryonic development of this animal. First, we comprehensively listed up regulatory genes, i.e. genes encoding sequence-specific transcription factors and signaling molecules, and then determined their expression patterns from the 1-cell stage to the late tailbud stage. We comprehensively knocked-down regulatory genes expressed in the early embryos, and reconstructed the regulatory networks among them. Recently we performed chromatin immunoprecipitation on 11 core transcription factors important for endomesoderm specification. We found that 58 of the 76 interactions are direct. We also identified the tightly interconnected cis-regulatory networks. We experimentally validated the network connections by overexpression of three transcription factors. The regulatory networks we revealed also gave us many implications that remained to be resolved. One of them is the function of ADMP (anti-dorsalizing morphogenetic protein), which belongs to a BMP family. As in the Xenopus embryo, this protein was shown to be involved in the dorsoventral patterning of the embryo, but the detailed mechanism was not necessarily unsolved. We again surveyed the genome for candidate genes involved in the BMP-signaling under a less strict condition. As a result, we identified a novel antagonist specific for ADMP. This antagonist played an essential role in the dorso-ventral patterning of the tailbud embryo. We also found that these two functional related genes, ADMP and its antagonist, were related to each other at the level of transcription. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Evolution of the micromeres in echinoids Takuya Minokawa Research Center for Marine Biology, Tohoku University The formation of “micromere quartet” in the vegetal-most region of the 16-cell-stage embryo is one of the common embryonic features of Euechinoidea (a group of modern echinoids). Two different functions of the micromeres and its descendants have been reported. First, they differentiate autonomously into skeletogenic mesenchyme cells. Second, the micromeres and its descendants send signals to induce endomesoderm in neighboring cells. These functions have established and modified during the divergence of echinoids. We study various echinoid model systems (the “primitive” Cidaroidea echinoid Prionocidaris baculosa, the “derived” direct developing echinoid Peronella japonica, and others) to gain an understanding of the molecular and cellular mechanisms that underlie the evolution of micromere specification and functions. The comparative study between Cidaroidea and Euechinoidea allows us to estimate the ancestral mode of the larval skeletogenic mechanism. The 16-cell-stage Prionocidaris embryos do not form “micromere quartet”. The expression patterns of several skeletogenesis-related genes in Prionocidaris are different from that of the Euechinoidea orthologs and resemble to that of the starfish orthologs, suggesting that Prionocidaris preserves the ancestral mechanism for larval skeletogenesis of the echinoid. The comparison between the indirect- and the direct-developing echinoids allows us to study the mechanical basis of the evolution of a novel developmental program. The micromeres in Peronella embryo do not express the inductive signals for endomesoderm. Several endomesoderm specification genes in Peronella show modified expression patterns. Based on these comparative studies, the evolution of micromeres and its function will be discussed. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Pattern formation in two dimensions Stanislav Y. Shvartsman Departments of Chemical and Biological Engineering and Molecular Biology; Lewis-Sigler Institute for Integrative Genomics, Princeton University. During Drosophila oogenesis, a gradient of Epidermal Growth Factor Receptor (EGFR) activation patterns the follicle cells, which form an epithelium over the developing oocyte. In response to their patterning by EGFR, follicle cells acquire a number of different fates necessary for the formation of the eggshell, a complex structure that protects the embryo and mediates its interaction with the environment. Genetic studies of Drosophila oogenesis established it as a premiere model for the mechanistic analysis of developmental signaling [1]. Based on the results of these studies, we are using the EGFR-patterning of the follicle cells to establish systems-level models of pattern formation by locally activated signaling pathways. We have quantified the gradient of EGFR activation in the follicle cells, identified dozens of its target genes, and begun to explore mechanisms of their regulation [2-7]. Analysis of the expression patterns of these genes suggests that they obey a combinatorial code, based on a small number of building blocks and geometric operations (Figure 1). The building blocks in this code are related to the distributions of inductive signals that pattern the follicular epithelium. The geometric operations can be potentially explained by the Boolean operations at the cis-regulatory modules of the EGFR target genes. Our recent experiments identified some of these modules, providing support for the combinatorial code hypothesis. 1. 2. 3. 4. 5. 6. 7. Berg, C.A. (2005). The Drosophila shell game: patterning genes and morphological change. Trends Genet 21, 346-355. Goentoro, L.A., Reeves, G.T., Kowal, C.P., Martinelli, L., Schupbach, T., and Shvartsman, S.Y. (2006). Quantifying the Gurken morphogen gradient in Drosophila oogenesis. Dev Cell 11, 263-272. Yakoby, N., Bristow, C.A., Gong, D., Schafer, X., Lembong, J., Zartman, J.J., Halfon, M.S., Schüpbach, T., and Shvartsman, S.Y. (2008). A combinatorial code for pattern formation in Drosophila oogenesis. Dev Cell 15, 725-737. Lembong, J., Yakoby, N., and Shvartsman, S.Y. (2009). Pattern formation by dynamically interacting network motifs. Proc Natl Acad Sci 106, 3213-3218. Yakoby, N., Lembong, J., Schupbach, T., and Shvartsman, S.Y. (2008). Drosophila eggshell is patterned by sequential action of feedforward and feedback loops. Development 135, 343-351. Zartman, J.J., Kanodia, J.S., Cheung, L.S., and Shvartsman, S.Y. (2009). Feedback control of the EGFR signaling gradient: superposition of domain splitting events in Drosophila oogenesis. Development 136, 2903-2911. Zartman, J.J., Cheung, L.S., Niepielko, M., Bonini, C., Haley, B., Yakoby, N., and Shvartsman, S.Y. (2011). Pattern formation by a moving morphogen source. Physical Biology 8, 045003. Figure 1: Complex patterns can be constructed from a small number of building blocks and geometric operations. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Evolving regulatory gene repertoire Shigehiro Kuraku Department of Biology, University of Konstanz, Germany Decades of molecular developmental studies have led to discoveries of regulatory networks governing various morphogenetic processes. Cross-species comparisons among diverse animals have revealed a high level of conservation of gene repertoires responsible for those developmental processes. A prevalent idea is that changes in regulations or functions of commonly shared genes have resulted in phenotypic diversity, through heterotopy, heterochrony, or co-option. This idea, which was established in the pre-genomic era, should now be verified with whole genome sequences of diverse organisms. My team has been interested in detecting losses of developmental genes of vertebrates in well-studied gene families. Our bioinformatic survey has revealed quite a few genes that mark taxon-specific gains or losses. They included the Bmp16 gene, closely related to Bmp2 and Bmp4 and missing in mammalian and bird genomes, as well as the Pax4 gene, closely related to Pax6 and missing in the currently available chicken and Xenopus genome sequences. We also scrutinized vanishing members of the Hox clusters, Hox14 genes, absent in the genomes of all traditional model vertebrates. These data, together with results from genome-wide analyses, emphasize a more dynamic nature of developmental gene repertoire in vertebrates than documented before. The approaches employed here, based on molecular phylogenetics and genome informatics, should potentially address more questions in life science regarding the laws of genome organization and expression in model organisms as well as us humans. Generation of neural primordia in vertebrate embryos by mechanisms that challenge the classical models Hisato Kondoh Graduate School of Frontier Biosciences, Osaka University The textbook view holds that segregation of the three germ layers (ectoderm, mesoderm, endoderm) is the process specifying cell lineages in early embryos. Our study challenges this, by showing that paraxial mesoderm (which gives muscle and bone) and posterior neural plate (spinal cord precursor) are in fact derived from common bi-potential precursors “axial stem cells” in the caudal lateral epiblast. This requires Tbx6 (a mesoderm-dedicated regulator) to turn off Sox2 (a neural regulator). Because of this, Tbx6-null mouse embryos develop ectopic neural tubes at the expense of paraxial mesoderm. Cell type specification is therefore separate from germ layer formation. Our study further shows that anterior neural plate (brain precursor) is derived directly from the epiblast (embryonic blastoderm). The mechanisms to derive neural plate differ depending on the embryonic axial levels. These findings will impact on contemporary stem cell research, where the regulation of cell lineage specification is the major issue. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Fins-to-Limbs Transition: Apical fold morphogenesis and mesenchymal cell differentiation Tohru Yano, Hiroki Yoshihara and Koji Tamura Graduate School of Life Sciences, Tohoku University One of morphological aspects of fins-to-limbs transition is loss of a fin-specific structure, fin ray, which includes back-to-back sheets of epidermis (apical fold, AF) and membrane bones (lepidotrichia). Studies suggesting similarities between fins and limbs have revealed shared mechanisms underlying their development, but little is known about development of the finspecific structure. We studied some developmental features of the pectoral fin ray, focusing on AF morphogenesis/function and developmental origin of the fin ray mesenchyme. We found that the AF can anatomically and molecularly be divided into the proximal part (pAF) and the distal part (dAF) and that the dAF regulates AF outgrowth at later stages of fin development. Interestingly, removal of the whole AF did not result in fin truncation unlike AER removal in tetrapod limbs but gave rise to reformation of the AER and resultant elongation of the endosekeletal region. Fin skeletons can be categorized into endochondral bones (endoskeletal elements) and membrane bones (fin ray elements). Endochondral bones in the pectoral fin correspond to limb skeletal elements in tetrapods that developmentally originate from the lateral plate mesoderm (LPM). On the other hand, the developmental origin of fin ray bones, fin-specific elements, remains unclear, although there are some arguments that the membrane bones are derived from neural crest cells migrating into the fins. Our cell lineage tracing with some labeling methods, however, provided evidence that those mesenchymal cells in the pectoral fin also originate from the LPM. Taken together, our results suggest that in pectoral fin development, fin ray mesenchymal cells from the LPM migrate into a space between back-to-back sheets of pAF and differentiate into bones through intramembranous ossification. Distal elongation of the AF enables this migration and resultant fin ray formation, and failure of transformation from the AER to AF might therefore have been an important event for fins-to-limbs transition during tetrapod evolution. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Origin of evolutionay novelties in turtles by developmental repatterning Shigeru Kuratani Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology (CDB) The turtle carapace, or the dorsal moiety of the shell, is derived from the rib, and is often regarded to represent a typical evolutionary novelty for its reversed topographical relationship with the pectoral girdle that is situated within the shell, unlike in other amniotes. To reveal the developmental factors behind this anatomical discrepancy, comparative molecular analyses of Pelodiscus sinensis embryos were carried out. During P. sinensis development, basic topographic relationships between muscle- and skeletal anlagen were largely conserved among amniotes, but some muscles that initially arose in the limb bud acquired novel insertion unique to the turtles. Thus the turtle body plan appears to be based upon novel direction of folding as well as invention of new connectivity. Along the line of turtle-specific folding arises the turtle-specific embryonic structure called the carapacial ridge (CR), which has been suspected to induce the turtle-specific rib growth pattern. To explore the mechanical background for this folding, we isolated CRABP-1, Sp-5, Lef-1, and APCDD1 as CR-specific genes. These genes were not P. sinensis-specific, but the evolutionary changes were introduced to ther regulation uniquley in the lineage of turtles. Inactivation of Lef-1 function or surgical removal of CR lead to the partial arrest of marginal growth of the carapace, suggesting that this structure is primarily for the carapacial growth, not for the rib patterning. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Hierarchical interactions that regulate craniofacial development and evolution Rich Schneider Department of Orthopaedic Surgery, University of California at San Francisco Craniofacial development is a highly dynamic and hierarchical process that involves multiple gene regulatory networks, distinct embryonic lineages, and reciprocal signaling interactions among cells and tissues. Mechanisms that orchestrate the various aspects of this complex process and ultimately enable the neural, skeletal, muscular, vascular, and epidermal components of the head to become structurally and functionally integrated, remain unclear. Our research focuses on the extent to which one progenitor population, the neural crest, serves as a primary source of patterning information during craniofacial development. Cranial neural crest cells originate along the dorsal margins of the neural tube, and they migrate extensively throughout the head. Their derivatives include dermis, cartilage, bone, and muscular connective tissues, and they interact extensively with non-neural crest-derived elements such as blood vessels, muscles, epidermis, and nerves. To test the regulatory abilities of the neural crest, we have established an experimental chimeric system using two distinct avian species, quail and duck. This approach exploits the fact that embryonic quail and duck are morphologically distinct and have considerably different rates of maturation. We transplant pre-migratory neural crest cells between quail and duck embryos, which challenges resultant chimeras to assimilate donor versus host-specific differences in growth and morphology. We find that within quailduck chimeras, donor neural crest cells execute autonomous molecular programs and regulate gene expression in adjacent host tissues. This in turn, establishes the timing of histogenesis, and affects the size, shape, and location of anatomical structures derived from both the donor and the host. Thus, neural crest cells function as a primary source of spatiotemporal patterning information during craniofacial development, and in this capacity have played an essential role in facilitating morphological change during the course of evolution. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Advantage of sea urchin embryos for the analysis of GRN Koji Akasaka Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo Sea urchin embryos have made great contribution to the study of gene regulatory network (GNR) in metazoans. The availability of huge numbers of synchronized embryos, sufficient amounts of nuclei extract to use for purification of transcription factors, and the use of an in vivo transcription system using developing embryonic cells have all shown that sea urchin embryos are extraordinary animals for research. Furthermore, morpholino antisense oligonucleotides have accelerated the study of GRN in sea urchin embryos. Taking advantage of superior properties of sea urchin embryos, we analyzed mechanism of regulation of gene expression of arylsulfatase (Ars), which serves as a model gene for study of spatial and temporal gene expression. The major enhancer element of Ars composed of the Otx target sequence and CAAT sequences is located in the 1st intron. Otx binding sites alone have little effect on the activity of an Ars promoter, but when both Otx binding sites and CAAT sequences are present in the enhancer region of Ars, the DNA fragment shows a high enhancer activity. A gel mobility shift assay reveals that Otx and CAAT box binding proteins bind to the enhancer. The activation domain of Otx resides in the C terminal region. The N-terminal region is responsible for the enhancement of transactivation of the Ars promoter, although that region itself does not function as an activation domain. These findings suggest that Otx regulates Ars by interacting with different co-factors in sea urchin development. Screening of target genes using morpholino antisense oligonucleotides is a powerful method for analysis of GRN; although, these findings suggest that black box still remains poised between the regulatory gene and the target gene. In order to uncover the black box, analysis of coordination with different transcription factors and interactions of these transcription factors with the cis-regulatory elements is required. Experimental system using sea urchin embryos should contribute to clarification of the molecular mechanisms of regulation of gene expression and contribute to the network diagram of the GNR. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Morphogenetic movements are driven by task-specific transcription factor control David R. McClay, Department of Biology, Duke University, Durham, NC 27708 The epithelial-mesenchymal transition (EMT) of skeletogenic cells in the sea urchin embryo occurs at 9 hrs after fertilization. A comprehensive developmental gene regulatory network is known that describes the specification of those cells. Using that network as a guide, each transcription factor in the network was selectively perturbed and then the EMT monitored. It was learned that one transcription factor cassette controls the de-adhesion component of EMT, another controls motility, and still other cassettes control endocytosis, exocytosis, polarity, invasion, and directed cell shape change. The transcription factors involved are used later for a subsequent EMT and may be essential for each EMT event in the embryo. Other morphogenetic events such as invagination of the archenteron, movement of the primordial germ cells into the coelomic pouches, formation and patterning of the mesodermal structures each involve components of the upstream specification gene regulatory network that controls the cell biology of the morphogenetic event. The goal is to define the control devices and discover how they control the morphogenetic movements. In each case some of the upstream transcription factors are known and some of their endpoint targets are known but the full system of each morphogenetic movement is complex and involves a number of network subcircuits, most of which have yet to be discovered. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) The origin and evolution of the endoderm gene regulatory network Athula H. Wikramanayake Department of Biology, University of Miami, Coral Gables, FL 33146 Gastrulation is a uniquely metazoan process, and the evolution of this character was arguably one of the pivotal events that drove the diversification of the metazoa. Segregation of germ layers and the onset of gastrulation movements in ancient embryos were likely preceded by the assembly and activation of a gene regulatory network (GRN) that specified a regulatory state for endoderm at one pole of ancient embryos. How this primordial GRN was assembled and activated in ancient embryos is not known, but GRN theory and comparative molecular embryology studies are providing unique insight into this process. Theory predicts that those GRNs that are activated during early development, and function to specify broad regional identities form kernels that are conserved over vast evolutionary periods due to the catastrophic consequences of losing any single link in the network. Hence, identification of endoderm GRN kernels conserved between bilaterians, and outgroups to the bilateria such as the cnidaria will likely provide key insight into the GRNs that shaped early animal evolution. In the sea urchin the nuclear entry of ß-catenin, a key signal transducer in the canonical Wnt pathway, is a critical upstream input into the now well-described endoderm GRN kernel. While endoderm GRN kernels have not been as extensively characterized in other bilaterians, nuclear ß-catenin marks the site of gastrulation in embryos of several bilaterian taxa, and in many cases, it has been shown that signaling via this protein is crucial for endoderm specification. In most bilaterians, the site of gastrulation is established predictably with respect to the animal-vegetal (AV) axis, and in general, these embryos develop endoderm from vegetal-half blastomeres. In contrast to bilaterians, cnidarians form endoderm at the animal pole. We have shown that nuclear ß-catenin signaling in blastomeres at the animal pole is essential for endoderm specification in this taxon indicating that a ß-catenin-dependent endoderm GRN kernel specified endoderm in the last common ancestor to bilaterians and cnidarians over 600 mya. We have proposed that the site of endoderm specification was moved from the animal pole of the last common ancestor to cnidarians and bilaterians, to the vegetal pole of the bilaterian last common ancestor. This radical shift in the site of endoderm GRN activation may have been enabled by the transfer of a key upstream activator of the endoderm kernel from the animal pole to the vegetal pole, thus resulting in the in toto shift of this GRN kernel in the urbilaterian. This event may have been a key trigger for the evolution of the bilaterian body plan and the subsequent radiation of this clade. This work is supported by a grant from the National Science Foundation, USA to AHW. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Development, genomic, and regeneration studies of a crinoid, Oxycomanthus japonicus Mariko Kondo Misaki Marine Biological Station and Center for Marine Biology, The University of Tokyo Among the echinoderms, sea urchins and sea stars have been studied as model organisms. Crinoids (sea lilies and feather stars) seem to be the least familiar group of the echinoderms, since most of them occur in the deep sea. Nevertheless, crinoids are potentially important model organisms for research, since they are considered the group of animals that have diverged the earliest from the other extant echinoderms. The feather star, Oxycomanthus japonicus, can be collected in shallower waters and maintained in cages hung in the cove of our station. This enables us to use the feather star for research, namely, on evolution and development of the echinoderm body plan and on regeneration. Given the very unique body plan of echinoderms, expression and genomic organization of Hox genes in echinoderms has been of great interest. To see whether Hox genes reflect the individual unique body plans observed in echinoderms, we are using the feather star for comparative and evolutionary studies. It has been reported that, unlike the well-known Hox gene clusters of vertebrates, the sea urchin Hox cluster lacks several Hox gene members and other modifications are present. Several of the sea urchin Hox genes are expressed along the oral-aboral axis during a certain period of early embryogenesis. From the feather star, our group has so far identified nine Hox genes and analyzed their expression during embryogenesis. To elucidate the genomic characteristics of the feather star Hox cluste, we are working on genomic BAC and fosmid clones to show the alignment and orientation of these genes. Our data indicate the presence of a single Hox cluster. Echinoderms, with the exception of sea urchins, exhibit a strong capacity for regeneration. Feather stars also are reported to regenerate most of its body parts. In natural conditions, their arms autotomize quite frequently, and this is followed by regeneration. There have been morphological or histological studies on crinoid regeneration, but only few have dealt with the molecular basis of regeneration. We have isolated several genes from the feather star. We are currently analyzing the expression of these genes during regeneration to test their involvement. Home Program Registration Speakers/Abstract Organizers Poster session Contact Special lecture 特別講演 (日本語) Gene regulatory network involved in the notochord formation in the Ciona intestinalis embryo Nori Satoh Marine Genomics Unit, Okinawa Institute of Science and Technology The notochord is a most prominent feature of chordates. An elucidation of molecular mechanisms involved in the formation of notochord in ascidian (urochordate) embryos is therefore essential for better understanding not only of its differentiation mechanism but also the origin and evolution of chordates. The ascidian larva develops the notochord that consists of exactly 40 cells, of which lineage is completely documented. A member of T-box family transcription factor genes, Brachyury, is a key regulator for the notochord formation. Its zygotic expression commences at the 64-cell stage when A-line cells are destined to give rise to only notochord. The upstream cascade leading to Ci-Bra (Brachyury of Ciona intestinalis) includes maternal b-catenin and p53, and zygotic FoxD, FGF and ZicL. The downstream cascade of CiBra includes ~400 genes involved in the notochord formation. We wish to discuss the complexity of gene regulatory network responsible for the notochord formation in ascidian embryos.