Kaoru S. Imai

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.

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.

Fgf genes in the basal chordate Ciona intestinalis.

Abstract. In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.

Region specific gene expressions in the central nervous system of the ascidian embryo.

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway.

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.

macho-1-related genes in Ciona embryos

Abstract. In ascidians, maternal factor(s) localized in the myoplasm of the egg are essential for specification and subsequent differentiation of larval muscle cells. The macho-1 gene of Halocynthia roretzi encodes a zinc-finger protein: the gene is only expressed maternally, the resultant maternal mRNA is localized to the myoplasm, and the gene function is essential for muscle cell differentiation. Here we have characterized macho-1 homologues, Ci-macho1 of Ciona intestinalis and Cs-macho1 of Ciona savignyi. Interestingly, we found that the Cionamacho-1 genes are expressed both maternally and zygotically: their maternal transcript is localized to the myoplasm while their zygotic expression is seen after neurulation in cells of the central nervous system. Functional suppression of Cs-macho1 with morpholino antisense oligonucleotide resulted in inhibition of the initiation of zygotic expression of a muscle-specific actin gene. We propose a possible evolutionary scenario in which an ancestral Zic-related gene gave rise to both the macho-1-like muscle determinant gene as well as neuronal Zic genes.

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. Here, we determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, we found that most of the previously determined interactions are direct. We focused on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. We also found many interactions that had not been predicted by simple gene knockdown experiments, and we showed that a significant fraction of TF-DNA interactions make major contributions to the regulatory control of target gene expression.

RETRACTED: Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.