Characterization of a maternal T-Box gene in Ciona intestinalis.

A new T-box gene, CiVegTR, was isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago.

Brachyury downstream notochord differentiation in the ascidian embryo.

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 Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation.

Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo.

Expression of the Brachyury (Ci-Bra) gene of the ascidian Ciona intestinalis is initiated at the 64-cell stage. Gene expression is restricted to notochord precursor cells, and Ci-Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci-Bra-downstream genes that are expressed in notochord cells were isolated. The present determination, by whole-mount in situ hybridization, of the temporal expression patterns of 19 notochord-specific and 20 notochord-predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord-specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

Lineage-specific regulation of the Ciona snail gene in the embryonic mesoderm and neuroectoderm.

The snail gene encodes a highly conserved, zinc-finger transcription factor that has been implicated in the specification of mesodermal and neuronal tissues in a variety of organisms. In the ascidian, Ciona intestinalis, snail (Ci-sna) is expressed at the 32-cell stage in derivatives of the B4.1 blastomere, including B6.2, B6.4, and B7.5, which give rise to the primary-lineage tail muscles of the tadpole. At later stages, Ci-sna is expressed in the lineages that will form the secondary tail muscle, the lateral ependymal cells of the spinal cord, and the dorsal cells of the cerebral vesicle. A minimal, 504-bp cis-regulatory sequence from the Ci-sna promoter region, the B4.1 enhancer, is shown to direct the expression of heterologous promoters in primary-lineage muscles. Furthermore, evidence is presented that cis-regulatory elements necessary for expression in both the secondary muscle and neuronal lineages are separate from the B4.1 enhancer. We discuss the possibility that the classical muscle determinant present in ascidian eggs may correspond to bHLH activators, which bind to specific E-box sequences contained in the B4.1 enhancer.

Dorsoventral patterning of the vertebrate neural tube is conserved in a protochordate.

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. Here we analyze the expression of HNF-3beta and snail homologues in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. A combination of in situ hybridization and promoter fusion analyses was used to demonstrate that the Ciona HNF-3beta homologue is expressed in the ventralmost ependymal cells of the neural tube, while the Ciona snail homologue is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. We propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians.