The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains ϳ16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Tunicates are the only animals that perform cellulose biosynthesis. The tunicate gene for cellulose synthase, Ci-CesA, was likely acquired by horizontal transfer from bacteria and was a key innovation in the evolution of tunicates. Transposon-based mutagenesis in an ascidian, Ciona intestinalis, has generated a mutant, swimming juvenile (sj). Ci-CesA is the gene responsible for the sj mutant, in which a drastic reduction in cellulose was observed in the tunic. Furthermore, during metamorphosis, which in ascidians convert the vertebrate-like larva into a sessile filter feeder, sj showed abnormalities in the order of metamorphic events. In normal larvae, the metamorphic events in the trunk region are initiated after tail resorption. In contrast, sj mutant larvae initiated the metamorphic events in the trunk without tail resorption. Thus, sj larvae show a ''swimming juvenile'' phenotype, the juvenile-like trunk structure with a complete tail and the ability to swim. It is likely that ascidian cellulose synthase is required for the coordination of the metamorphic events in the trunk and tail in addition to cellulose biosynthesis.Minos ͉ swimming juvenile ͉ Ci-CesA ͉ metamorphosis T unicates are the only animals able to perform cellulose biosynthesis (1-3). Cellulose synthase is an enzyme that has a central role in cellulose biosynthesis; the gene (Ci-CesA) for cellulose synthase is encoded in the Ciona intestinalis genome and expressed in the larval ectoderm (4). Recent molecular phylogenetic analyses of Ci-CesA and Cs-CesA of Ciona savignyi suggest that the gene has been acquired by horizontal transfer from bacteria (5, 6). Ascidian cellulose synthase may be involved in the formation of the tunic, the cellulose-containing structure that surrounds the surface of the body to protect against predators. However, functional analysis showing that ascidian cellulose synthase contributes to the tunic formation in vivo has not been reported.Mutant analysis is a powerful way to understand gene function. In an ascidian, C. savignyi, mutagenesis with a chemical mutagen N-ethyl-N-nitrosourea, successfully revealed the function of prickle (7) and tyrosinase (8). Recently, germ-line transgenesis has been achieved in two ascidians, C. intestinalis and C. savignyi, with a Tc1͞mariner superfamily transposon Minos (9-14). The insertion of Minos into the Ciona genome indicates that Minos can be used as a mutagen, because Minos disrupts the gene function if it is inserted into a genomic region that encodes a gene.Here, we have isolated a mutant, swimming juvenile (sj), which was caused by an insertion of Minos into the C. intestinalis genome. sj shows defects in the tunic structure of swimming larvae. In addition to the tunic phenotype, they showed an abnormal order of metamorphic events, suggesting that the regulation of the timing of these events is affected in sj mutants. Ci-CesA is the gene responsible for the sj mutant, and the drastic loss of cellulose was observed in the tunic of sj mutants. These results indicate that ascidian ...

The tadpole larva of the basal chordate Ciona intestinalis has the most simplified, basic body-plan of chordates. Because it has a compact genome with a complete draft sequence, a large quantity of EST͞cDNA information, and a short generation time, Ciona is a suitable model for future genetics. We establish here a transgenic technique in Ciona that uses the Tc1͞mariner superfamily transposon Minos. Minos was integrated efficiently into the genome of germ cells and transmitted stably to subsequent generations. In addition, an enhancer-trap line was obtained. This is a demonstration of efficient, Minos-mediated transgenesis in marine invertebrates.ascidian ͉ transgenic technique ͉ enhancer trap

Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types - larval tail muscle, adult body-wall muscle and heart - expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.

A virtual image showing ascidian Ciona intestinalis larvae swimming to "corals" located at the bottom of the picture. On top right is a tailbud embryo still in the chorion before hatching. Swimming larvae are carrying constructs driving the expression of green fluorescent proteins in epidermis. Larvae become juveniles through metamorphosis (lower right). Juveniles and adult neural complex pictures derive from a transgenic line with Ror‐b regulatory sequences, which drives yellow fluorescent protein expression at the top of endostyles in juveniles and at the margins of the so‐called ciliated funnels in neural complexes. The corals were reconstructed from details of two adult neural gland complexes artificially merged. Our ascidian facilities in closed‐systems contribute to the maintenance of such transgenic lines. See Joly et al., Developmental Dynamics 236:1832–1840.