A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt,TGFbeta, Hedgehog and JAK/STAT signaling pathways

Cell-cell interactions play important roles in a variety of developmental processes, and therefore molecules involved in the signaling pathways have been studied extensively. Recently, the draft genome sequence of the basal chordate, Ciona intestinalis, was determined. Here we annotated genes for the signaling pathways of Wnt, transforming growth factor beta (TGFbeta), Hedgehog, and JAK/STAT in the genome of Ciona intestinalis. The Ciona genome contains ten wnt genes, six frizzled genes, four sFRP genes, ten TGFbeta family member genes, five TGFbeta-receptor genes, and five Smad genes; most of the genes were found with less redundancy than in vertebrate genomes. The other genes in the signaling pathways are present as a single copy in the Ciona genome. In addition, all of the identified genes for the signaling pathway, except for a few genes, have EST evidence, and their cDNAs are available from the Ciona intestinalis gene collection. Therefore, Ciona intestinalis may provide an experimental system for exploring the basic genetic cascade associated with the signaling pathways in chordates.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.     

The Notch signaling pathway in the cnidarian Hydra

Many of the major pathways that govern early development in higher animals have been identified in cnidarians, including the Wnt, TGFbeta and tyrosine kinase signaling pathways. We show here that Notch signaling is also conserved in these early metazoans. We describe the Hydra Notch receptor (HvNotch) and provide evidence for the conservation of the Notch signaling mode via regulated intramembrane proteolysis. We observed that nuclear translocation of the Notch intracellular domain (NID) was inhibited by the synthetic gamma-secretase inhibitor DAPT. Moreover, DAPT treatment of hydra polyps caused distinct differentiation defects in their interstitial stem cell lineage. Nerve cell differentiation proceeded normally but post-mitotic nematocyte differentiation was dramatically reduced. Early female germ cell differentiation was inhibited before exit from mitosis. From these results we conclude that gamma-secretase activity and presumably Notch signaling are required to control differentiation events in the interstitial cell lineage of Hydra.     

Control and integration of cell signaling pathways during C. Elegans vulval development

Vulval development in the Caenorhabditis elegans hermaphrodite represents a simple, genetically tractable system for studying how cell signaling events control cell fata decisions. Current models suggest that proper specification of vulval cell fates relies on the integration of multiple signaling systems, including one that involves a receptor tyrosine kinase (RTK)→Ras→mitogen activated protein kinase (MAPK) cascade and one that involves a LIN-12/Notch family receptor. In this review, we first discuss how genetic strategies are being used to identify and analyze components that control vulval cell fate decisions. We then describe the different signaling systems that have been elucidated and how they relate to one another. Finally, we highlight several recently characterized genes that encode positive regulators, negative regulators or potential targets of the RTK→Ras→MAPK cascade involved in vulval induction.     

miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway

The nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyury-regulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3'UTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development.     

Three insulin-relaxin-like genes in Ciona intestinalis

The Ciona intestinalis genome harbors three insulin-like genes: INS-L1, -L2 and -L3. Conserved synteny between the Ciona-human genomes predicts that Ciona INS-Ls are orthologous to the vertebrate insulin-relaxin family, but this relation cannot be inferred from molecular phylogeny. A conserved protein core with six cysteines; typical arrangement of B-, C- and A-protein domains; pro-protein maturation mode; and putative insulin receptor-binding sites were identified in Ciona INS-L proteins. ESTs used to assemble exonic sequences of INS-Ls combined with qRT-PCR analysis provided evidence that the predicted genes are expressed in the developing and adult Ciona. Our results support that Ciona INS-L1 is orthologous to the vertebrate insulin-like/relaxin genes, INS-L2 to insulin genes and INS-L3 to IGF genes. Our analysis also implies that the insulin-like/relaxin ancestor switched receptor type from tyrosine kinase- to GPCR-type, whereas insulin-IGF subfamily retained the tyrosine kinase-type of receptor. We propose that this receptor-switch occurred after the time when urochordates branched from the common chordate lineage, but before the two genome-duplications at the root of the vertebrates.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VII. Molecules involved in the regulation of cell polarity and actin dynamics

In the present study, genes involved in the pathways that establish cell polarity and cascades regulating actin dynamics were identified in the completely sequenced genome of Ciona intestinalis, a basal chordate. It was revealed that the Ciona genome contains orthologous genes of each component of aPKC-Par and PCP pathways and WASP/WAVE/SCAR and ADF/cofilin cascades, with less redundancy than the vertebrate genomes, suggesting that the conserved pathways/cascades function in Ciona development. In addition, the present study found that the orthologous proteins of five gene groups (Tc10, WRCH, RhoD, PLC-L, and PSKH) are conserved in humans and Ciona but not in Drosophila melanogaster, suggesting a similarity in the gene composition of Ciona to that of vertebrates. Ciona intestinalis, therefore, may provide refined clues for the study of vertebrate development and evolution.     

A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway.     

The little R cell that could

Drosophila eye development provides an excellent model system to study the role of inter-cellular signaling in the specification of unique cell fates. Behavioral screens by Benzer and his colleagues led to the identification of a gene, Sevenless, a receptor tyrosine kinase (RTK) receptor, required for the specification of the UV sensitive R7 cell. Genetic analysis further showed that the Ras/Raf/MAPK pathway function downstream of Sevenless in the specification of R7 fate. Signaling mediated by another RTK, EGFR and Notch have also been shown to function in either an antagonistic or a synergistic manner in the specification of cell fate during eye development. In some instances, these pathways are linked in a sequential manner by the regulation of the expression of Notch ligand, Delta by EGFR, while in others, these pathways function in a combinatorial fashion on enhancer elements to control target gene expression. In this review, we highlight the elegant genetic strategies used by several laboratories in early elucidation of the Sevenless pathway which helped link the RTK receptor to the Ras/Raf/MAPK cascade and discuss how EGFR and Notch signaling pathways are used in a reiterative manner and by combining in different modes, generate sufficient diversity required for the specification of unique cell fates.     

Unusual number and genomic organization of Hox genes in the tunicate Ciona intestinalis

Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage.     

A genome-wide survey of genes for enzymes involved in pigment synthesis in an ascidian, Ciona intestinalis

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix

Cell junctions and the extracellular matrix (ECM) are crucial components in intercellular communication. These systems are thought to have become highly diversified during the course of vertebrate evolution. In the present study, we have examined whether the ancestral chordate already had such vertebrate systems for intercellular communication, for which we have searched the genome of the ascidian Ciona intestinalis. From this molecular perspective, the Ciona genome contains genes that encode protein components of tight junctions, hemidesmosomes and connexin-based gap junctions, as well as of adherens junctions and focal adhesions, but it does not have those for desmosomes. The latter omission is curious, and the ascidian type-I cadherins may represent an ancestral form of the vertebrate type-I cadherins and desmosomal cadherins, while Ci-Plakin may represent an ancestral protein of the vertebrate desmoplakins and plectins. If this is the case, then ascidians may have retained ancestral desmosome-like structures, as suggested by previous electron-microscopic observations. In addition, ECM genes that have been regarded as vertebrate-specific were also found in the Ciona genome. These results suggest that the last common ancestor shared by ascidians and vertebrates, the ancestor of the entire chordate clade, had essentially the same systems of cell junctions as those in extant vertebrates. However, the number of such genes for each family in the Ciona genome is far smaller than that in vertebrate genomes. In vertebrates these ancestral cell junctions appear to have evolved into more diverse, and possibly more complex, forms, compared with those in their urochordate siblings.     

Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway.     

Large-scale characterization of genes specific to the larval nervous system in the ascidian Ciona intestinalis

The central and peripheral nervous systems (CNS and PNS) of the ascidian tadpole larva are comparatively simple, consisting of only about 350 cells. However, studies of the expression of neural patterning genes have demonstrated overall similarity between the ascidian CNS and the vertebrate CNS, suggesting that the ascidian CNS is sufficiently complex to be relevant to those of vertebrates. Recent progress in the Ciona intestinalis genome project and cDNA project together with considerable EST information has made Ciona an ideal model for investigating molecular mechanisms underlying the formation and function of the chordate nervous system. Here, we characterized 56 genes specific to the nervous system by determining their full-length cDNA sequences and confirming their spatial expression patterns. These genes included those that function in the nervous systems of other animals, especially those involved in photoreceptor-mediated signaling and neurotransmitter release. Thus, the nervous system-specific genes in Ciona larvae will provide not only probes for determining their function but also clues for exploring the complex network of nervous system-specific genes.