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.

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.

Ascidians, belonging to the subphylum Urochordata, the earliest branch from the lineage to the vertebrates, exhibit a prototypical morphogenesis of chordates in the larval development, although they subsequently metamorphose into adults with a unique body structure. Recent draft genome analysis of the ascidian Ciona intestinalis has identified 9 Hox genes, which, however, have been located on five scaffolds. Similarly, expression patterns of Ciona Hox genes are largely unknown, although some data have been available for a few Hox member genes. Thus, the cluster structure and colinearity of Hox genes are still an enigma in C. intestinalis. To address these issues, we used fluorescence in situ hybridization and whole-mount in situ hybridization techniques and examined the genomic organization and spatiotemporal expression of all Hox as well as extended Hox member genes (Evx and Mox) of C. intestinalis. We found that seven of nine Ciona Hox genes are located on a single chromosome with some ordering exceptions, and the other genes, including Evx and Mox, are located on three other chromosomes. Some Ciona Hox genes, if not all, exhibited spatially coordinated expression within the larval central nervous system and the gut of the juvenile. In light of these observations, we suggest that the cluster organization and colinearity of the Hox genes are under dispersing and disintegrating conditions in C. intestinalis.

Comparative studies on expression patterns of developmental genes along the anterior-posterior axis of the embryonic central nervous system (CNS) between vertebrates and ascidians led to the notion of "tripartite organization," a common ground plan of the CNS, consisting of the anterior, central and posterior regions expressing Otx, Pax2/5/8 and Hox genes, respectively. In ascidians, however, descriptions and interpretations about expression of the developmental genes regarded as region specific have become not necessarily consistent. To address this issue, we examined detailed expression of key developmental genes for the ascidian CNS, including Otx, Pax2/5/8a, En, Fgf8/17/18, Dmbx, Lhx3 and Hox genes, in the CNS around the junction of the trunk and tail of three different tailbud-stage embryos of Ciona intestinalis, employing double-fluorescence in situ hybridization, followed by staining with DAPI to precisely locate expressing cells for each gene. Based on these observations, we have constructed detailed gene expression maps of the region at the tailbud stages. Our analysis shows that expression of several genes regarded as markers for specific domains in the ascidian CNS changes dynamically within a relatively short period. This motivates us to revisit to the tripartite ground plan and the origin of the midbrain-hindbrain boundary (MHB) region.