Yutaka Satou

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets (‘amphioxus’) are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic ∼520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. II. Genes for homeobox transcription factors.

Homeobox-containing genes play crucial roles in various developmental processes, including body-plan specification, pattern formation and cell-type specification. The present study searched the draft genome sequence and cDNA/EST database of the basal chordate Ciona intestinalis to identify 83 homeobox-containing genes in this animal. This number of homeobox genes in the Ciona genome is smaller than that in the Caenorhabditis elegans, Drosophila melanogaster, human and mouse genomes. Of the 83 genes, 76 have possible human orthologues and 7 may be unique to Ciona. The ascidian homeobox genes were classified into 11 classes, including Hox class, NK class, Paired class, POU class, LIM class, TALE class, SIX class, Prox class, Cut class, ZFH class and HNF1 class, according to the classification scheme devised for known homeobox genes. As to the Hox cluster, the Ciona genome contains single copies of each of the paralogous groups, suggesting that there is a single Hox cluster, if any, but genes orthologous to Hox7, 8, 9 and 11 were not found in the genome. In addition, loss of genes had occurred independently in the Ciona lineage and was noticed in Gbx of the EHGbox subclass, Sax, NK3, Vax and vent of the NK class, Cart, Og9, Anf and Mix of the Paired class, POU-I, III, V and VI of the POU class, Lhx6/7 of the LIM class, TGIF of the TALE class, Cux and SATB of the Cut class, and ZFH1 of the ZFH class, which might have reduced the number of Ciona homeobox genes. Interestingly, one of the newly identified Ciona intestinalis genes and its vertebrate counterparts constitute a novel subclass of HNF1 class homeobox genes. Furthermore, evidence for the gene structures and expression of 54 of the 83 homeobox genes was provided by analysis of ESTs, suggesting that cDNAs for these 54 genes are available. The present data thus reveal the repertoire of homeodomain-containing transcription factors in the Ciona genome, which will be useful for future research on the development and evolution of chordates.

Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks

Achieving a real understanding of animal development obviously requires a comprehensive rather than partial identification of the genes working in each developmental process. Recent decoding of genome sequences will enable us to perform such studies. An ascidian, Ciona intestinalis, one of the animals whose genome has been sequenced, is a chordate sharing a basic body plan with vertebrates, although its genome contains less paralogs than are usually seen in vertebrates. In the present study, we discuss the genomewide approach to networks of developmental genes in Ciona embryos. We focus on transcription factor genes and some major groups of signal transduction genes. These genes are comprehensively listed and examined with regard to their embryonic expression by in situ hybridization (http://ghost.zool.kyoto-u.ac.jp/tfst.html). The results revealed that 74% of the transcription factor genes are expressed maternally and that 56% of the genes are zygotically expressed during embryogenesis. Of these, 34% of the transcription factor genes are expressed both maternally and zygotically. The number of zygotically expressed transcription factor genes increases gradually during embryogenesis. As an example, and taking advantage of this comprehensive description of gene expression profiles, we identified transcription factor genes and signal transduction genes that are expressed at the early gastrula stage and that work downstream of β-catenin, FoxD and/or Fgf9/16/20. Because these three genes are essential for ascidian endomesoderm specification, transcription factor genes and signal transduction genes involved in each of the downstream processes can be deduced comprehensively using the present approach.

Regulatory Blueprint for a Chordate Embryo

Ciona is an emerging model system for elucidating gene networks in development. Comprehensive in situ hybridization assays have identified 76 regulatory genes with localized expression patterns in the early embryo, at the time when naïve blastomeres are determined to follow specific cell fates. Systematic gene disruption assays provided more than 3000 combinations of gene expression profiles in mutant backgrounds. Deduced gene circuit diagrams describing the formation of larval tissues were computationally visualized. These diagrams constitute a blueprint for the Ciona embryo and provide a foundation for understanding the evolutionary origins of the chordate body plan.

Axial patterning in cephalochordates and the evolution of the organizer.

The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.

Ciona intestinalis: an emerging model for whole-genome analyses

Abstract The tadpole larvae of the ascidian Ciona intestinalis possess the most simplified chordate body plan. Analysis of the Ciona draft genome indicates that the ∼153–159Mb genome contains ∼16 000 protein-coding genes. Among these is a fundamental set of conserved chordate proteins involved in cell signaling and development. A thorough examination of Ciona gene expression (the transcriptome) is ongoing, including large-scale expressed sequence tag analyses, cDNA sequencing and in situ hybridization screens. Together with recent advances in the methodology used to investigate gene regulation and function, these make Ciona an attractive experimental system for genome level analysis of chordate developmental genetics.

An Integrated Database of the Ascidian, Ciona intestinalis: Towards Functional Genomics

Abstract An integrated genome database is essential for future studies of functional genomics. In this study, we update cDNA and genomic resources of the ascidian, Ciona intestinalis, and provide an integrated database of the genomic and cDNA data by extending a database published previously. The updated resources include over 190,000 ESTs (672,396 in total together with the previous ESTs) and over 1,000 full-insert sequences (6,773 in total). In addition, results of mapping information of the determined scaffolds onto chromosomes, ESTs from a full-length enriched cDNA library for indication of precise 5′-ends of genes, and comparisons of SNPs and indels among different individuals are integrated into this database, all of these results being reported recently. These advances continue to increase the utility of Ciona intestinalis as a model organism whilst the integrated database will be useful for researchers in comparative and evolutionary genomics.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. I: Genes for bHLH transcription factors

The basic helix-loop-helix (bHLH) proteins are transcription factors that play important roles in many biological processes, including the development of various animals. We identified 46 genes encoding bHLH proteins in the draft genome sequence of the basal chordate Ciona intestinalis. These 46 genes represent an almost complete set of bHLH genes in this animal. This number is comparable to 39 and 58 bHLH genes in those of Caenorhabditis elegans and Drosophila melanogaster but is much smaller than the 125 in the human genome. Genes that encode NeuroD, Beta3, Oligo, SCL, NSCL, SRC, Clock and Bmal were not found in the Ciona genome. We could also identify groucho and CBP orthologues in the Ciona genome. In addition, the comparison between the numbers of orthologous human and Ciona bHLH factors reveals the simplicity of the Ciona genome, in accordance with its phylogenetic position within chordates. The present analysis provides an insight into the basic set of bHLH genes that the last common ancestor of ascidians and vertebrates had, and will provide important background information for future studies using ascidians as a model system for vertebrate development.

The ascidian Mesp gene specifies heart precursor cells

Understanding the molecular basis of heart development is an important research area, because malformation of the cardiovascular system is among the most frequent inborn defects. Although recent research has identified molecules responsible for heart morphogenesis in vertebrates, the initial specification of heart progenitors has not been well characterized. Ascidians provide an appropriate experimental system for exploring this specification mechanism, because the lineage for the juvenile heart is well characterized, with B7.5 cells at the 110-cell stage giving rise to embryonic trunk ventral cells (TVCs) or the juvenile heart progenitors. Here, we show that Cs-Mesp, the sole ortholog of vertebrate Mesp genes in the ascidian Ciona savignyi, is specifically and transiently expressed in the embryonic heart progenitor cells (B7.5 cells). Cs-Mesp is essential for the specification of heart precursor cells, in which Nkx, HAND and HAND-like (NoTrlc) genes are expressed. As a result, knockdown of Cs-Mesp with specific morpholino antisense oligonucleotides causes failure of the development of the juvenile heart. Together with previous evidence obtained in mice, the present results suggest that a mechanism for heart specification beginning with Mesp through Nkx and HAND is conserved among chordates.