Ancient intron insertion sites and palindromic genomic duplication evolutionally shapes an elementally functioning membrane protein family.

BACKGROUND: In spite of the recent accumulation of genomic data, the evolutionary pathway in the individual genes of present-day living taxa is still elusive for most genes. Among ion channels, inward K+ rectifier (IRK) channels are the fundamental and well-defined protein group. We analyzed the genomic structures of this group and compared them among a phylogenetically wide range with our sequenced Halocynthia roretzi, a tunicate, IRK genomic genes. RESULTS: A total of 131 IRK genomic genes were analyzed. The phylogenic trees of amino acid sequences revealed a clear diversification of deuterostomic IRKs from protostomic IRKs and suggested that the tunicate IRKs are possibly representatives of the descendants of ancestor forms of three major groups of IRKs in the vertebrate. However, the exon-intron structures of the tunicate IRK genomes showed considerable similarities to those of Caenorhabditis. In the vertebrate clade, the members in each major group increased at least four times those in the tunicate by various types of global gene duplication. The generation of some major groups was inferred to be due to anti-tandem (palindromic) duplication in early history. The intron insertion points greatly decreased during the evolution of the vertebrates, remaining as a unique conservation of an intron insertion site in the portion of protein-protein interaction within the coding regions of all vertebrate G-protein-activated IRK genes. CONCLUSION: From the genomic survey of a family of IRK genes, it was suggested that the ancient intron insertion sites and the unique palindromic genomic duplication evolutionally shaped this membrane protein family.

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants.

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.

Cleavage-arrested cell triplets from ascidian embryo differentiate into three cell types depending on cell combination and contact timing.

During early ascidian development, which is a prototype of early vertebrate development, anterior neuroectoderm cells (a4.2) from the eight-cell embryo are destined to become anterior neural structures including the brain vesicle, while presumptive notochordal neural cells (A4.1) become larval posterior neural structures including motoneurons. Whereas, an anterior quadrant cell (A3) of the four-cell embryo, from which both anterior neuroectoderm (a4.2) and notochordal neural cells (A4.1) are derived, has both fates. Cleavage-arrested cell triplets were prepared from the anterior quadrant cell and a pair of anterior neuroectoderm cells (A3-aa triplet) or a pair of presumptive notochordal neural cells (A3-AA triplet), and cultured in contact. Differentiation of cells in the triplet was determined electrophysiologically by observing cell type-specific currents. In the A3-aa triplet, when two neuroectoderm cells and an anterior quadrant cell were prepared from the same batch of embryos, all three cells in the triplet developed into neuronal cells in 60 % of cases, but in 40 % of cases all of them differentiated into epidermal cells. However, when the batch of embryos from which neuroectoderm cells were prepared was fertilized 3 h later than that from which the anterior quadrant cell was prepared all three cells in the triplet consistently became neuronal cells. In contrast, when the batch of embryos from which neuroectoderm cells were prepared was fertilized 3 h earlier, all three cells became epidermal. In the A3-AA triplet no switching of differentiation occurred and all three cells in the triplet differentiated into neuronal cells, although the amplitude of inward current was often small. In neuralized A3-aa triplets the spikes in the anterior quadrant cell were characteristically small in amplitude and brief in duration, suggesting the presence of A-currents, which is a characteristic feature of posterior neuronal differentiation. In contrast, the spikes in the anterior neuroectoderm cells were large in amplitude and long in duration, chracteristic to the anterior neuronal type. The majority of single isolated anterior quadrant cells became non-excitable. However, the minority was apparently autonomously neuralized to become the posterior neuronal type. In neuralized A3-AA triplets, the majority of anterior quadrant cells was induced to become the anterior neuronal type. When isolated anterior quadrant cells were neuralized with subtilisin, a protease, they also predominantly became the anterior neuronal type. While, in medium containing a fibroblast growth factor posterior neuralization of isolated anterior quadrant cells was facilitated, but the anterior neuronal type, although minor, appeared anew. These observations indicate that the multiple fates of the anterior quadrant cell expressed in vivo were effectively reproduced in this experimental condition at the single cell level. Interactive differentiation in this triplet system recapitulates not only fundamental neural induction of ascidian neuroectoderm cells, but also functional and positional specificity within the neuronal group.