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.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential CiBra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Received March 22, 1999; revised version accepted May 3, 1999. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain (Herrmann et al. 1990;Kispert et al. 1995;Conlon et al. 1996). In vertebrates, Brachyury is initially expressed throughout the presumptive mesoderm, and during later stages the expression pattern is gradually restricted to the developing notochord and tailbud. Brachyury notochord differentiation is essential in all vertebrates that have been studied, including mice, frogs, and zebrafish (for review, see Herrmann and Kispert 1994;Smith 1997;Papaioannou and Silver 1998).Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, Halocynthia roretzi (Yasuo and Satoh 1993) and Ciona intestinalis (Corbo et al. 1997a). The spatial and temporal patterns of the gene expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm (Nakatani and Nishida 1994). Overexpression of the Halocynthia Brachyury gene (As-T) via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage (Yasuo and Satoh 1998). In addition, misexpression of As-T also causes transformation of endoderm and neuronal lineages into notochord (Yasuo and Satoh 1998). These results indicate that the ascidian Brachyury gene is a critical determinant of the notochord. Here we report that the misexpression of the Brachyury gene (Ci-Bra) of C. intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra overexpression. We isolated and characterized 38 different notochord-specific genes that may include potential targets of the ascidian Brachyury. Results and DiscussionThe fork head/HNF-3␤ gene of C. intestinalis (Ci-fkh) is expressed in the endoderm, endodermal strand, notochord, and ventral ependymal cells of the neural tube (Corbo et al. 1997b). A 2.6-kb genomic DNA fragment from the 5Ј-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into one-cell embryos (Fig. 1A). The Ci-Bra gene was misexpr...

What Is an Ascidian?Ascidians, or sea squirts, are invertebrate chordates that belong to the earliest branch in the chordate phylum, the subphylum Urochordata or Tunicata (Figure 1A). Ascidian larvae possess a prototypical chordate body plan

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.

Thanks to their transparent and rapidly developing mosaic embryos, ascidians (or sea squirts) have been a model system for embryological studies for over a century. Recently, ascidians have entered the postgenomic era, with the sequencing of the Ciona intestinalis genome and the accumulation of molecular resources that rival those available for fruit flies and mice. One strength of ascidians as a model system is their close similarity to vertebrates. Literature reporting molecular homologies between vertebrate and ascidian tissues has flourished over the past 15 years, since the first ascidian genes were cloned. However, it should not be forgotten that ascidians diverged from the lineage leading to vertebrates over 500 million years ago. Here, we review the main similarities and differences so far identified, at the molecular level, between ascidian and vertebrate tissues and discuss the evolution of the compact ascidian genome.