Noriyuki Satoh

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.

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. I. Up to the eight-cell stage.

Cell lineages during development of ascidian embryos were analyzed by injection of horseradish peroxidase as a tracer enzyme into identified cells at the one-, two-, four-, and eight-cell stages of the ascidians, Halocynthia roretzi, Ciona intestinalis, and Ascidia ahodori. Identical results were obtained with eggs of the three different species examined. The first cleavage furrow coincided with the bilateral symmetry plane of the embryo. The second furrow did not always divided the embryo into anterior and posterior halves as each of the anterior and posterior cell pairs gave rise to different tissues according to their destinies, which became more definitive in the cell pairs at the eight-cell stage. Of the blastomeres constituting the eight-cell stage embryo, the a4.2 pair (the anterior animal blastomeres) differentiated into epidermis, brain, and presumably sense organ and palps. Every descendant cell of the b4.2 pair (the posterior animal blastomeres) has been thought to become epidermis; however, the horseradish peroxidase injection probe revealed that the b4.2 pair gave rise to not only epidermis but also muscle cells at the caudal tip region of the developing tailbud-stage embryos. The A4.1 pair (the anterior vegetal blastomeres) developed into endoderm, notochord, brain stem, spinal cord, and also muscle cells next the caudal tip muscle cells. From the B4.1 pair (the posterior vegetal blastomeres) originated muscle cells of the anterior and middle parts of the tail, mesenchyme, endoderm, endodermal strand, and also notochord at the caudal tip region. These results clearly demonstrate that muscle cells are derived not only from the B4.1 pair, as has hitherto been believed, but also from both the b4.2 and A4.1 pairs.

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.

Chasing tails in ascidians: developmental insights into the origin and evolution of chordates

The ascidian tadpole larva is regarded as a prototype of the ancestral chordate. Here we consider recent studies on the development of the tadpole larva that provide new insights into chordate origins and evolution. The notochord of ascidian larvae and vertebrates appear to be homologous structures based on their induction by endoderm and expression of the Brachyury (T) gene. The muscle cells of ascidian larvae also appear homologous to those of vertebrates based on their expression of bHLH myogenic and muscle-type actin genes, although they are specified by cytoplasmic determinants localized in the egg as well as embryonic induction. Studies of the tailless larvae of anural ascidians have resulted in the identification of Manx, a gene that may control tail development and evolution. These and other results support the ascidian tadpole prototype for the ancestral chordate.

An Ascidian Homolog of the Mouse Brachyury (T) Gene is Expressed Exclusively in Notochord Cells at the Fate Restricted Stage

Ascidians are primitive chordates in which a well‐organized notochord is formed in the tail of the tadpole larva. The Brachyury (T) gene in the mouse is essential for formation of mesoderm and, in particular, of notochord. We report here the expression of an ascidian homolog (As‐T) of the mouse T gene. A cDNA clone for the As‐T gene contains a single open reading frame that encodes a polypeptide of 471 amino acids. Although the overall degree of amino acid identity was not very high (39.9%, ascidian/mouse), in the N‐terminal half the extent of amino acid identity was 78.5% (ascidian/mouse). The expression of As‐T was transient. A single band corresponding to a 2.2 kb mRNA was first detected at the 64‐cell stage, and a distinct band was found at the gastrula stage, at which time accumulation of the transcripts was maximal. The transcript was barely detectable at the larval stage and was undetectable in adult tissues. The occurrence of As‐T transcripts was restricted to notochord‐lineage cells, and no other cell types expressed the As‐T gene. In addition, the timing of As‐T expression coincided with the time of restriction of developmental fate exclusively to notochord in the notochord‐lineage cells. It is likely that the Brachyury gene was present when the ancestors of chordates with a notochord emerged and that the primary function of this gene is to specify notochord cells.

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. II. The 16- and 32-cell stages.

Cell lineages during development of ascidian embryos were analyzed by injecting horseradish peroxidase as a tracer enzyme into identified cells of the 16-cell and 32-cell stage embryos of Halocynthia roretzi. Most of the blastomeres of these embryos developed more kinds of tissues than have hitherto been reported, and therefore, the developmental fates of each blastomere are more complex. It has been thought that every blastomere of the 64-cell stage ascidian embryo gives rise to only one kind of tissues, but the finding that the several blastomeres at the 32-cell stage developed into at least three different kinds of tissues, clearly indicates that the stage at which the fates of every blastomere are determined to one tissue is later than the 64-cell stage. The results also clearly demonstrate that muscle cells are derived not only from B-line cells (B5.1, B5.2, B6.3, and B6.4) but also from A-line cells (A5.2 and A6.4) and b-line cells (b5.3 and b6.5). Based on the present analysis as well as other studies, complete cell lineages of muscle cells up to their terminal differentiation have been proposed. In addition, lineages of nervous system, notochord, and epidermis are also discussed.

Novel pattern of Brachyury gene expression in hemichordate embryos

Together with echinoderms and chordates, hemichordates constitute the third major group of the deuterostomes, which share a number of common developmental features. The Brachyury gene is responsible for the formation of notochord, the most defining feature of chordates. Therefore, isolation and characterization of the hemichordate homolog of Brachyury is key to understand the origin and evolution of chordates. Here we show that the hemichordate Brachyury gene (PfBra) is expressed in two regions of the gastrula and young tornaria larva, the archenteron invagination region and the stomodeum invagination region.

Timing of initiation of muscle specific gene expression in the ascidian embryo precedes that of developmental fate restriction in lineage cells

The lineage of muscle cells in the ascidian embryo is well documented. Of the B‐line blastomeres, B7.4 cells become restricted to give rise to muscle at the 44‐cell stage, whereas B7.8 and B7.5 cells become restricted to muscle at the 64‐cell stage. In this study we addressed three issues concerning the timing of initiation of muscle‐specific structural gene transcription: (i) whether the initiation of the transcription of muscle‐specific genes is correlated to that of developmental fate restriction; (ii) if there is a relationship in the timing, whether it is applicable to B7.4‐, B7.5‐ and B7.8‐sublineages; and (iii) whether two muscle‐specific genes, one for actin and the other for the myosin heavy chain, show the same pattern of temporal expression.

Phylogenetic Relationships between Solitary and Colonial Ascidians, as Inferred from the Sequence of the Central Region of their Respective 18S rDNAs

Ascidians (tunicates) are primitive chordates. In spite of their elevated phylogenetic position in the animal kingdom, ascidians have evolved a varied reproductive repertoire; some of them live as individuals (solitary ascidians), while others form colonies (colonial ascidians). Colonial ascidians propagate asexually by budding and strobilation, and they have an extensive capacity for regeneration. However, the orthodox taxonomic classification of ascidians categorizes them into two major groups (the orders Enterogona and Pleurogona), irrespective of their solitary or colonial life style. To examine whether the orthodox classification of ascidians is substantiated by molecular phylogeny, the complete nucleotide sequence of a region of about 1000 base pairs in the central part of their respective 18S rDNAs was determined, and the sequences were compared among five solitary and three colonial ascidians. The phylogenetic tree deduced from these results suggests that the three species of Enterogona and the five species of Pleurogona examined form discrete and separate groups irrespective of their potential to form colonies. Therefore, a solitary or colonial life style is likely to have developed independently after the divergence of the two major groups of ascidians.

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.