Hidetoshi Saiga

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.

Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi

Hrlim is a LIM class homeobox gene that was first isolated from the ascidian Halocynthia roretzi. To assess its roles in early development of the ascidian, spatial and temporal expression of Hrlim was examined by whole mount in situ hybridization. This revealed that transcription of Hrlim is activated at the 32-cell stage specifically in the endoderm lineage. Hrlim is also transiently expressed in all notochord precursor cells. Expression in the endoderm lineage continues through to the middle of gastrulation. After gastrulation, Hrlim is expressed in certain lineages that give rise to subsets of cells in the brain and spinal cord. Based on these observations, it is suggested that Hrlim plays multiple distinct roles in ascidian embryogenesis.

Hroth, an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis

To obtain insight into the axis-forming mechanism in ascidian embryogenesis, Hroth, an ascidian counterpart of orthodenticle/otx, was isolated from Halocynthia roretzi and its expression in embryogenesis was examined by whole mount in situ hybridization. It was revealed that Hroth is expressed in both involuting mesoendoderm and anterior ectoderm during gastrulation while later expression is restricted to the sensory vesicle and anterior epidermis. Expression pattern of Hroth around gastrulation was compared with that of Hrlim, the ascidian LIM class homeobox gene that is known to be expressed during gastrulation. In the light of the present findings on the expression of Hroth, properties of the axis-forming mechanism in ascidian embryogenesis are discussed.

Early specification of intestinal epithelium in the chicken embryo: a study on the localization and regulation of CdxA expression.

CdxA, a chicken homeobox‐containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA‐expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial‐mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.

Origin of Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and MASP-3 Involved in the Lectin Complement Pathway Traced Back to the Invertebrate, Amphioxus

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.

Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis, which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, we compared in both species the regulation of Otx, a gene with a shared complex expression pattern. We found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved, in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. We propose that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates.

HrNodal, the ascidian nodal-related gene, is expressed in the left side of the epidermis, and lies upstream of HrPitx.

Abstract. The nodal-related genes are well known for their fundamental roles during vertebrate development, including mesoderm induction, neural induction, and left-right axis formation, as several nodal-related genes show left-sided expression in mesodermal lineages. We have isolated the first non-vertebrate nodal-related gene, HrNodal, from the ascidian Halocynthia roretzi. During the late cleavage and gastrula stages, HrNodal is transiently and bilaterally expressed in several different cell lineages. Expression at the tailbud stage is observed asymmetrically in the left side, but unexpectedly only in the epidermis of the embryo. We also demonstrate the relationship of HrNodal with HrPitx, a Halocynthia homologue of the Pitx2 gene. HrNodal overexpression results in the disturbance of left-sided HrPitx expression. Our results demonstrate that left-right specification during ascidian embryogenesis involves the HrNodal gene, and that the left-sidedness of the expression is evolutionarily conserved throughout the chordate clade. Electronic Supplementary Material is available if you access this article at http://dx.doi.org/10.1007/s00427-002-0242-3. On that page (frame on the left side), a link takes you directly to the supplementary material.

Comprehensive survey and classification of homeobox genes in the genome of amphioxus, Branchiostoma floridae.

The homeobox genes comprise a large and diverse gene superfamily, many of which encode transcription factors with pivotal roles in the embryonic development of animals. We searched the assembled draft genome sequence of an amphioxus, Branchiostoma floridae, for genes possessing homeobox sequences. Phylogenetic analysis was used to divide these into gene families and classes. The 133 amphioxus homeobox genes comprise 60 ANTP class genes, 29 PRD genes (excluding Pon and Pax1/9), nine TALE genes, seven POU genes, seven LIM genes, five ZF genes, four CUT genes, four HNF genes, three SINE genes, one CERS gene, one PROS gene, and three unclassified genes. Ten of the 11 homeobox gene classes are less diverse in amphioxus than humans, as a result of gene duplication on the vertebrate lineage. Amphioxus possesses at least one member for all of the 96 homeobox gene families inferred to be present in the common ancestor of chordates, including representatives of the Msxlx, Bari, Abox, Nk7, Ro, and Repo gene families that have been lost from tunicates and vertebrates. We find duplication of several homeobox genes in the cephalochordate lineage (Mnx, Evx, Emx, Vent, Nk1, Nedx, Uncx, Lhx2/9, Hmbox, Pou3, and Irx) and several divergent genes that probably originated by extensive sequence divergence (Hx, Ankx, Lcx, Acut, Atale, Azfh, Ahbx, Muxa, Muxb, Aprd1–6, and Ahnf). The analysis reveals not only the repertoire of amphioxus homeobox genes but also gives insight into the evolution of chordate homeobox genes.

Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos

An extracellular signaling molecule acts on several types of cells, evoking characteristic and different responses depending on intrinsic factors in the signal-receiving cells. In ascidian embryos, notochord and mesenchyme are induced in the anterior and posterior margins, respectively, of the vegetal hemisphere by the same FGF signal emanating from endoderm precursors. The difference in the responsiveness depends on the inheritance of the posterior-vegetal egg cytoplasm. We show that macho-1, first identified as a localized muscle determinant, is also required for mesenchyme induction, and that it plays a role in making the cell response differ between notochord and mesenchyme induction. A zygotic event involving snail expression downstream of maternal macho-1 mediates the suppression of notochord induction in mesenchyme precursors.