Michio Ogasawara

Ascidian homologs of mammalian thyroid peroxidase genes are expressed in the thyroid-equivalent region of the endostyle

The endostyle is a pharyngeal organ for the internal filter feeding of urochordates, cephalochordates, and larval lamprey. This organ is also considered to be homologous to the follicular thyroid gland of higher vertebrates. Thyroglobulin (Tg) and thyroid peroxidase (TPO) are specifically expressed in the thyroid gland of higher vertebrates, and they play an important role in iodine metabolism for the synthesis of thyroid hormones. Previous histochemical observations showed that iodine-concentrating and peroxidase activities were detected in zones 7, 8, and 9 of the ascidian endostyle, suggesting that these zones contains cells that are equivalent to those in the vertebrate follicular thyroid. In order to investigate the molecular developmental mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the thyroid gland, in the present study, we isolated and characterized cDNA clones for TPO genes, CiTPO from Ciona intestinalis and HrTPO from Halocynthia roretzi. Northern blot and in situ hybridization analyses revealed that the expression of the ascidian TPO genes was restricted to zone 7, one of the elements equivalent to the thyroid. These results provide the first evidence at the gene expression level for shared function between a part of the ascidian endostyle and the vertebrate follicular thyroid gland. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:158-169, 1999.

Gene expression profiles in young adult Ciona intestinalis

Abstract. Comparison of 12,230 expressed sequence tags (ESTs) of 3′ ends of cDNA clones derived from young adults of Ciona intestinalis allowed us to categorize them into 976 independent clusters. When the 5′-end sequences of 10,400 ESTs of the 976 clusters were compared with the sequences in databases, 406 of the clusters showed significant matches (P < E-15) with reported proteins with defined functions, while 117 showed matches with putative proteins for which there is not enough information to categorize their function, and 453 had no significant sequence similarities to known proteins. The 406 clusters with sequence similarity to proteins with defined functions consisted of 304 clusters related to proteins with functions common to many kinds of cells, 73 related to proteins associated with cell-cell communication and 29 related to transcription factors. Spatial expression of all of the 976 clusters was examined by a newly improved whole-mount in situ hybridization method. A total of 430 clusters did not show distinct in situ hybridization signals, while 122 clusters showed ubiquitous distribution of signals, and 253 clusters showed signals in multiple tissues. The remaining 171 clusters showed signals specific to a certain organ or tissue: 16 showed epidermis-specific expression, 3 were specific to the neural complex, 1 to heart, 6 to body-wall muscle, 94 to pharyngeal gill, 3 to esophagus, 26 to stomach, 1 to intestine and 21 to endostyle. Many of these organ-specific genes encode proteins with no sequence similarity to known proteins. The present analysis thus highlights characteristic gene expression profiles of Ciona young adults and provides not only molecular markers for organs and tissues but also transcriptomic information useful for further genomic analyses of this model organism.

Overlapping expression of amphioxus homologs of the thyroid transcription factor-1 gene and thyroid peroxidase gene in the endostyle: insight into evolution of the thyroid gland.

Abstract The endostyle is a pharyngeal organ of uro- chordates, cephalochordates, and primitive vertebrates. This organ has iodine-concentrating and iodine-metabolism activities, and therefore the endostyle is considered to be homologous to the follicle of the thyroid gland. In higher vertebrates the genes for both thyroid transcription factor-1 (TTF-1) and thyroid peroxidase (TPO) are expressed in the thyroid gland follicle. TTF-1 regulates the expression of TPO, which encodes an iodinating enzyme associated with thyroid hormone synthesis. A recent study showed that the ascidian TTF-1 and TPO genes are specifically expressed in the endostyle, but that the expression domains of these genes are not overlapping, suggesting that ascidian TPO is not regulated by TTF-1. To investigate the molecular mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the follicle of the thyroid gland, I isolated and characterized cDNA clones for the amphioxus homologs of the TTF-1 gene (BbTTF-1) and TPO gene (BbTPO) from Branchiostomabelcheri. Reverse transcriptase–polymerase chain reaction/Southern blotting revealed that both amphioxus TTF-1 and TPO genes are expressed mainly in the adult endostyle. Spatial and temporal expression patterns assessed by in situ hybridization revealed that BbTTF-1 is expressed in the endodermal cells during early embryogenesis and is maintained in all zones of the adult endostyle. On the other hand, expression of BbTPO is chiefly in zones 5 and 6 of the adult endostyle where it overlaps with that of BbTTF-1, and to a lesser extent in zones 1 and 3. This restriction of the expression of BbTTF-1 and BbTPO to the endostyle strongly suggests that the endostyle is homologous to the follicle of the thyroid gland. Moreover, the spatial and temporal expression patterns of these genes suggest that TTF-1 regulates TPO expression. The coexpression of these genes in amphioxus suggests that regulation of TPO by TTF-1 was present in the common ancestor of cephalochordates (acraniates) and craniates.

Tachykinin and Tachykinin Receptor of an Ascidian, Ciona intestinalis EVOLUTIONARY ORIGIN OF THE VERTEBRATE TACHYKININ FAMILY

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian γ-preprotachykinin A (γ-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH2 vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.

Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi.

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 (Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark (pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex.

Toll-like receptors of the ascidian Ciona intestinalis: prototypes with hybrid functionalities of vertebrate Toll-like receptors.

Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-κB induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNFα in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess “hybrid” biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition.

Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis

The vasopressin (VP)/oxytocin (OT) superfamily peptides are one of the most widely distributed neuropeptides and/or neurohypophysial hormones, but have ever not been characterized from any deuterostome invertebrates including protochordates, ascidians. In the present study, we show the identification of a novel VP/OT superfamily peptide and its receptor in the ascidian, Ciona intestinalis. Intriguingly, the Ciona VP/OT-related peptide (Ci-VP), unlike other 9-amino acid and C-terminally amidated VP/OT superfamily peptides, consists of 13 amino acids and lacks a C-terminal amidation. Mass spectrometry confirmed the presence of the 13-residue Ci-VP in the neural complex. Furthermore, 10 of 14 cysteines are conserved in the neurophysin domain, compared with other VP/OT counterparts. These results revealed that the VP/OT superfamily is conserved in ascidians, but the Ci-VP gene encodes an unprecedented VP/OT-related peptide and neurophysin protein. Ci-VP was also shown to activate its endogenous receptor, Ci-VP-R, at physiological concentrations, confirming the functionality of Ci-VP as an endogenous ligand. The Ci-VP gene was expressed exclusively in neurons of the brain, whereas the Ci-TK-R mRNA was distributed in various tissues including the neural complex, alimentary tract, gonad, and heart. These expression profiles suggest that Ci-VP, like other VP/OT superfamily peptides, serves as a multifunctional neuropeptides. Altogether, our data revealed both evolutionary conservation and specific divergence of the VP/OT superfamily in protochordates. This is the first molecular characterization of a VP/OT superfamily peptide and its cognate receptor from not only ascidians but also deuterostome invertebrates.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates.

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid‐like features such as iodine‐concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid‐related transcription factors such as TTF‐2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci‐FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid‐like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. Developmental Dynamics 233:1031–1037, 2005.

A Large-Scale Whole-Mount in situ Hybridization System: Rapid One-Tube Preparation of DIG-Labeled RNA Probes and High Throughput Hybridization using 96-Well Silent Screen Plates

Abstract Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes.

Calcitonin in a protochordate, Ciona intestinalis – the prototype of the vertebrate calcitonin/calcitonin gene-related peptide superfamily

The calcitonin (CT)/CT gene‐related peptides (CGRPs) constitute a large peptide family in vertebrates. However, no CT/CGRP superfamily members have so far been identified in invertebrates, and the evolutionary process leading to the diverse vertebrate CT/CGRP superfamily members remains unclear. In this study, we have identified an authentic invertebrate CT, Ci‐CT, in the ascidian Ciona intestinalis, which is the phylogenetically closest invertebrate chordate to vertebrates. The amino acid sequence of Ci‐CT was shown to display high similarity to those of vertebrate CTs and to share CT consensus motifs, including the N‐terminal circular region and C‐terminal amidated proline. Furthermore, the Ci‐CT gene was found to be the only Ciona CT/CGRP superfamily gene. Ci‐CT also exhibited less potent, but significant, activation of the human CT receptor, as compared with salmon CT. Physiological analysis revealed that Ci‐CT reduced the osteoclastic activity that is specific to vertebrate CTs. CD analysis demonstrated that Ci‐CT weakly forms an α‐helix structure. These results provide evidence that the CT/CGRP superfamily is essentially conserved in ascidians as well as in vertebrates, and indicate that Ci‐CT is a prototype of vertebrate CT/CGRP superfamily members. Moreover, expression analysis demonstrated that Ci‐CT is expressed in more organs than vertebrate CTs in the cognate organs, suggesting that an original CT/CGRP superfamily member gene was also expressed in multiple organs, and each CT/CGRP superfamily member acquired its current specific tissue distribution and physiological role concomitantly with diversification of the CT/CGRP superfamily during the evolution of chordates. This is the first report on a CT/CGRP superfamily member in invertebrates.