[Show abstract][Hide abstract] ABSTRACT: The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell-surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R-Bf-RAMP-LP complexes resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenmedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.
Full-text · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: More than 550 million years ago, chordates originated from a common ancestor shared with nonchordate deuterostomes by developing a novel type of larva, the "tadpole larva." The notochord is the supporting organ of the larval tail and the most prominent feature of chordates; indeed, phylum Chordata is named after this organ. In this review, we discuss the molecular mechanisms involved in the formation of the notochord over the course of chordate evolution with a special emphasis on a member of T-box gene family, Brachyury. Comparison of the decoded genome of a unicellular choanoflagellate with the genomes of sponge and cnidarians suggests that T-box gene family arose at the time of the evolution of multicellular animals. Gastrulation is a morphogenetic movement that is essential for the formation of two- or three-germ-layered embryos. Brachyury is transiently expressed in the blastopore (bp) region, where it confers on cells the ability to undergo invagination. This process is involved in the formation of the archenteron in all metazoans. This is a "primary" function of Brachyury. During the evolution of chordates, Brachyury gained an additional expression domain at the dorsal midline region of the bp. In this new expression domain, Brachyury served its "secondary" function, recruiting another set of target genes to form a dorsal axial organ, notochord. The Wnt/β-catenin, BMP/Nodal, and FGF-signaling pathways are involved in the transcriptional activation of Brachyury. We discuss the molecular mechanisms of Brachyury secondary function in the context of the dorsal-ventral (D-V) inversion theory and the aboral-dorsalization hypothesis. Although the scope of this review requires some degree of oversimplification of Brachyury function, it is beneficial to facilitate studies on the notochord formation, a central evolutionary developmental biology problem in the history of metazoan evolution, pointed out first by Alexander Kowalevsky.
No preview · Article · Feb 2012 · Evolution & Development
[Show abstract][Hide abstract] ABSTRACT: CIPRO database is an integrated protein database for a tunicate species Ciona intestinalis that belongs to the Urochordata. Although the CIPRO database deals with proteomic and transcriptomic data of a single species, the animal is considered unique in the evolutionary tree, representing a possible origin of the vertebrates and is a good model for understanding chordate evolution, including that of humans. Furthermore, C. intestinalis has been one of the favorites of developmental biologists; there exists a huge amount of accumulated knowledge on its development and morphology, in addition to the recent genome sequence and gene expression data. The CIPRO database is aimed at not only collecting published data, but also presenting unique information, including the unpublished transcriptomic and proteomic data and human curated annotation, for the use by researchers in broad research fields of biology and bioinformatics.
[Show abstract][Hide abstract] ABSTRACT: The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36,034 unique sequences, but for higher usability it covers 89,683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10,000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.
Full-text · Article · Nov 2010 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Brachyury plays a pivotal role in the notochord formation in ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was isolated as a gene downstream of Ci-Bra. This gene encodes a 307 amino-acid protein with a C-terminal phosphotyrosine interaction domain (PTB/PID). Expression of Ci-Noto4 commences at the neural plate stage and is specific to notochord cells. Suppression of Ci-Noto4 levels with specific antisense morpholino oligonucleotides resulted in the formation of two rows of notochord cells owing to a lack of midline intercalation between the bilateral populations of progenitor cells. In contrast, overexpression of Ci-Noto4 by injection of a Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized eggs disrupted the localization of notochord cells. Ci-Noto4 overexpression did not affect cellular differentiation in the notochord, muscle, mesenchyme, or nervous system. Analysis of Ci-Noto4 regions that are responsible for its function suggested significant roles for the PTB/PID and a central region, an area with no obvious sequence similarity to other known proteins. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline intercalation of notochord cells in chordate embryos.
Full-text · Article · Sep 2010 · The International journal of developmental biology
[Show abstract][Hide abstract] ABSTRACT: In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole-larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 9+2 axonemes were accompanied by a vesicle-containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle-containing fibrous mass and no 9+2 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic.
Preview · Article · Aug 2010 · Developmental Dynamics
[Show abstract][Hide abstract] ABSTRACT: Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified approximately 450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within approximately 3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region approximately 0.5-1.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates.
No preview · Article · Feb 2010 · ZOOLOGICAL SCIENCE
[Show abstract][Hide abstract] ABSTRACT: The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.
Full-text · Article · Feb 2009 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: CionaNotochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, Nf kappaB and TGF-beta signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates.
No preview · Article · Jan 2008 · Evolution & Development
[Show abstract][Hide abstract] ABSTRACT: The ascidian chordate Ciona intestinalis is an established model organism frequently exploited to examine cellular development and a rapidly emerging model organism with a strong potential for developmental systems biology studies. However, there is no standardized developmental table for this organism. In this study, we made the standard web-based image resource called FABA: Four-dimensional Ascidian Body Atlas including ascidian's three-dimensional (3D) and cross-sectional images through the developmental time course. These images were reconstructed from more than 3,000 high-resolution real images collected by confocal laser scanning microscopy (CLSM) at newly defined 26 distinct developmental stages (stages 1-26) from fertilized egg to hatching larva, which were grouped into six periods named the zygote, cleavage, gastrula, neurula, tailbud, and larva periods. Our data set will be helpful in standardizing developmental stages for morphology comparison as well as for providing the guideline for several functional studies of a body plan in chordate.
[Show abstract][Hide abstract] ABSTRACT: Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo.
Full-text · Article · Jul 2007 · Development Growth and Regeneration
[Show abstract][Hide abstract] ABSTRACT: T-box genes encode a family of transcription factors having conserved DNA-binding domains and diverged transcription regulatory domains, and each family member shows a specific expression pattern and plays a specific and crucial role in animal development. Two fundamental questions to be answered are whether the T-box gene functional specificity is located in the DNA-binding domain or in the transcription regulatory domain and how the specific expression of T-box genes is controlled. In the ascidian Halocynthia roretzi, Brachyury (HrBra) is expressed only in notochord cells while Tbx6 (HrTbx6) is expressed in muscle cells. In the present study, we made chimeric constructs of the two genes to determine the above mentioned questions. Our results suggest that the functional specificity of these two ascidian T-box genes is associated with the DNA-binding domain but not with the transcription regulatory domain. The 5' flanking region of both HrBra and HrTbx6 contains T-protein binding motifs near their minimal promoters that are associated with the autoregulative activation of these genes. Using the chimeric constructs, we also determined whether the autoregulative activity is mediated by the DNA-binding domain or by the transcription activation domain of the gene products. Our results suggest that the autoregulative activity of these two ascidian T-box genes is also mediated by the DNA-binding domain, not by the transcription activation domain of the encoded proteins.
Preview · Article · May 2005 · Development Growth and Regeneration
[Show abstract][Hide abstract] ABSTRACT: We previously reported that two NF-kappaB/Rel family members are involved in notochord formation of the ascidian Halocynthia roretzi. Here, we present evidence that the NF-kappaB/Rel signaling pathway plays important roles in the notochord formation in another ascidian, Ciona intestinalis. We first found that two NF-kappaB/Rel family members of C. intestinalis, Ci-rel1 and Ci-rel2, are splice variants: Ci-rel1 is a typical member, while Ci-rel2 is a C-terminally truncated short one. Ectopic expression of GFP-fusion proteins in the C. intestinalis notochord revealed that Ci-rel1 transiently moved into the nucleus in the initial tailbud stage, when concomitant expression of Ci-IkappaB, a C. intestinalis IkappaB homologue, was observed, indicating that Ci-rel1 is transiently activated in this stage. Ci-rel1, as well as Ci-rel2, is capable of binding to the kappaB sequence present upstream of Ci-IkappaB, suggesting that Ci-IkappaB is a target gene of Ci-rel1. Reporter gene assay suggests that the expression of Ci-IkappaB in the notochord is controlled by its kappaB sequence. Gene silencing of Ci-IkappaB by injection of the corresponding antisense morpholino oligonucleotide resulted in impairment of notochord formation in C. intestinalis, particularly in a defect in intercalation of notochord cells. Taken together, the results suggest that the regulation of Ci-rel1 by Ci-IkappaB, whose transcription is regulated by Ci-rel1, in the tailbud stage is essential for notochord formation in C. intestinalis.
Full-text · Article · Feb 2005 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: Members of the T-box family of transcription factors share an evolutionarily conserved DNA-binding domain and play significant roles in various processes of embryonic development. Vertebrate T-box genes are categorized into the following five major subfamilies (eight groups), depending on sequence similarities: Brachyury, Tbx1 (Tbx1/10, Tbx15/18/22, Tbx20), Tbx2/3/4/5 (Tbx2/3 and Tbx4/5), Tbx6, and Tbr/Eomes/TBX21. Ascidians are primitive chordates, and their tadpole larva are considered to represent the simplified and basic body plan of vertebrates. In addition, it has been revealed that the ascidian genome contains the basic ancestral complement of genes involved in development. The present characterization of cDNAs and survey of the Ciona intestinalis draft genome demonstrated that the Ciona genome contains a single copy gene for each of the Brachyury, Tbx1/10, Tbx15/18/22, Tbx20, Tbx2/3, and Tbr/Eomes/TBX21 groups, and at least three copies of the Tbx6 subfamily. Each of the Ciona T-box genes shows a characteristic expression pattern, although that of Tbx20 was not determined in the present study. These results provide basic information that will be useful for future studies of the function of each gene, genetic cascades of different T-box genes, and genome-wide surveys of evolutionary changes in the T-box gene structure and organization in this primitive chordate.
Preview · Article · Aug 2004 · Developmental Dynamics
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.
[Show abstract][Hide abstract] ABSTRACT: Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.