Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

scRNA-seq analysis of cells comprising the amphioxus notochord.

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

CatSper mediates not only chemotactic behavior but also the motility of ascidian sperm.

Introduction: Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration, and the sperm-specific Ca2+ channel CatSper has been shown to play an important role in the regulation of intracellular Ca2+. In particular, in mammals, CatSper is the only functional Ca2+ channel in the sperm, and mice deficient in the genes comprising the pore region of the Ca2+ channel are infertile due to the inhibition of sperm hyperactivation. CatSper is also thought to be involved in sea urchin chemotaxis. In contrast, in ascidian Ciona intestinalis, SAAF, a sperm attractant, interacts with Ca2+/ATPase, a Ca2+ pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is a functional Ca2+ channel in sperm. Results: We showed that CatSper is present in the sperm flagella of C. intestinalis as in mammalian species, although a small level of gene expression was found in other tissues. The spermatozoa of CatSper3 KO animals were significantly less motile, and some motile sperms did not show any chemotactic behavior. These results suggest that CatSper plays an important role in ascidians and mammals, and is involved in spermatogenesis and basic motility mechanisms.

Multi-regional expression of pancreas-related digestive enzyme genes in the intestinal chamber of the ascidian Ciona intestinalis type A.

Bilateria share sequential steps in their digestive systems, and digestion occurs in a pre-absorption step within a chamber-like structure. Previous studies on the ascidian Ciona intestinalis type A, an evolutionary research model of vertebrate organs, revealed that Ciona homologs of pancreas-related exocrine digestive enzymes (XDEs) are exclusively expressed in the chamber-like bulging stomach. In the development of the gastrointestinal tract, genes for the pancreas-related transcription factors, namely Ptf1a, Nr5a2, and Pdx, are expressed near the stomach. Recent organ/tissue RNA-seq studies on two Ciona species reported that transcripts of the XDE homologs exist in the intestinal regions, as well as in the stomach. In the present study, we investigated the spatial gene expression of XDE homologs in the gastrointestinal region of the C. intestinalis type A. Whole-mount in situ hybridization using adult and juvenile specimens revealed apparent expression signals of XDE homologs in a small number of gastrointestinal epithelial cells. Furthermore, two pancreas-related transcription factor genes, Nr5a2 and Pdx, exhibited multi-regional expression along the Ciona juvenile intestines. These results imply that ascidians may form multiple digestive regions corresponding to the vertebrate pancreas.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Voltage-sensing phosphatase (Vsp) regulates endocytosis-dependent nutrient absorption in chordate enterocytes.

Voltage-sensing phosphatase (Vsp) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. Vsp orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of Vsp in native tissues remains largely unknown. Here we report that zebrafish Vsp (Dr-Vsp), encoded by tpte gene, is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-Vsp-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-Vsp-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis Vsp (Ci-Vsp) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of Vsp in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species.

Differentiation of endostyle cells by Nkx2-1 and FoxE in the ascidian Ciona intestinalis type A: insights into shared gene regulation in glandular- and thyroid-equivalent elements of the chordate endostyle.

Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and ß-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle.

Nkx2-1 and FoxE regionalize glandular (mucus-producing) and thyroid-equivalent traits in the endostyle of the chordate Oikopleura dioica.

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis.

The emergence of nebulin repeats and evolution of lasp family proteins.

The LIM and SH3 domain protein (lasp) family, the smallest proteins in the nebulin superfamily, consists of vertebrate lasp-1 expressed in various non-muscle tissues, vertebrate lasp-2 expressed in the brain and cardiac muscle, and invertebrate lasp whose functions have been analyzed in Ascidiacea and Insecta. Gene evolution of the lasp family proteins was investigated by multiple alignments, comparison of gene structure, and synteny analyses in eukaryotes in which mRNA expression was confirmed. All invertebrates analyzed in this study belonging to the clade Filasterea, with the exception of Placozoa, have at least one lasp gene. The minimal actin-binding region (LIM domain and first nebulin repeat) and SH3 domain detected in vertebrate lasp-2 were found to be conserved among the lasp family proteins, and we showed that nematode lasp has actin-binding activity. The linker sequences vary among invertebrate lasp proteins, implying that the lasp family proteins have universal and diverse functions. Gene structures and syntenic analyses suggest that a gene fragment encoding two nebulin repeats and a linker emerged in Filasterea or Holozoa, and the first lasp gene was generated following combination of three gene fragments encoding the LIM domain, two nebulin repeats with a linker, and the SH3 domain.

An α7-related nicotinic acetylcholine receptor mediates the ciliary arrest response in pharyngeal gill slits of Ciona.

Ciliary movement is a fundamental process to support animal life, and the movement pattern may be altered in response to external stimuli under the control of nervous systems. Juvenile and adult ascidians have ciliary arrays around their pharyngeal gill slits (stigmata), and continuous beating is interrupted for seconds by mechanical stimuli on other parts of the body. Although it has been suggested that neural transmission to evoke ciliary arrest is cholinergic, its molecular basis has not yet been elucidated in detail. Here, we attempted to clarify the molecular mechanisms underlying this neurociliary transmission in the model ascidian Ciona Acetylcholinesterase histochemical staining showed strong signals on the laterodistal ciliated cells of stigmata, hereafter referred to as trapezial cells. The direct administration of acetylcholine (ACh) and other agonists of nicotinic ACh receptors (nAChRs) onto ciliated cells reliably evoked ciliary arrest that persisted for seconds in a dose-dependent manner. While the Ciona genome encodes ten nAChRs, only one of these called nAChR-A7/8-1, a relative of vertebrate α7 nAChRs, was found to be expressed by trapezial cells. Exogenously expressed nAChR-A7/8-1 on Xenopus oocytes responded to ACh and other agonists with consistent pharmacological traits to those observed in vivo Further efforts to examine signaling downstream of this receptor revealed that an inhibitor of phospholipase C (PLC) hampered ACh-induced ciliary arrest. We propose that homomeric α7-related nAChR-A7/8-1 mediates neurociliary transmission in Ciona stigmata to elicit persistent ciliary arrest by recruiting intracellular Ca2+ signaling.

Effect of Polycyclic Aromatic Hydrocarbons on Development of the Ascidian Ciona intestinalis Type A.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates; however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Cionaaryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.

Transcripts of the nebulin gene from Ciona heart and their implications for the evolution of nebulin family genes.

Nebulin is a 770 kDa protein that is localized along the thin filaments of skeletal muscles in vertebrates. It is also present in the striated muscles of Amphioxus, an invertebrate cephalochordate that is phylogenetically close to vertebrates. However, the nebulin of urochordate ascidians or its expression in invertebrate hearts has not been investigated. In this study, we investigated the structure and cardiac expression of the nebulin gene in Ciona intestinalis, a urochordate whose phylogeny lies between cephalochordates and vertebrates. As a result of the gene structure analysis, we found that the Ciona nebulin gene predicted to be 62 kb and consists of 143 exons. The nebulin was expected to consist of a unique N-terminal region, followed by 155 nebulin repeats, another unique region, a Ser-rich region and a C-terminal SH3 domain. Whole-mount in situ hybridization experiments showed that the Ciona nebulin gene was expressed in a variety of muscles, including hearts. However, Western blot analysis using antibody to Ciona nebulin did not detect the presence of full-length nebulin. Alternatively, RT-PCR experiments on samples of Ciona heart detected the expression of nebulette-like and nrap-like isoforms from the Ciona nebulin gene. These results indicate that, similarly to vertebrate hearts, Ciona hearts do not express nebulin, but rather nrap- and nebulette-like isoforms. These results also imply that the nebulin, nebulette and nrap genes in vertebrates were separated from an ancestral invertebrate nebulin gene during vertebrate evolution.

Molecular and evolutionary aspects of the protochordate digestive system.

The digestive system is a functional unit consisting of an endodermal tubular structure (alimentary canal) and accessory organs that function in nutrition processing in most triploblastic animals. Various morphologies and apparatuses are formed depending on the phylogenetical relationship and food habits of the specific species. Nutrition processing and morphogenesis of the alimentary canal and accessory organs have both been investigated in vertebrates, mainly humans and mammals. When attempting to understand the evolutionary processes that led to the vertebrate digestive system, however, it is useful to examine other chordates, specifically protochordates, which share fundamental functional and morphogenetic molecules with vertebrates, which also possess non-duplicated genomes. In protochordates, basic anatomical and physiological studies have mainly described the characteristic traits of suspension feeders. Recent progress in genome sequencing has allowed researchers to comprehensively detail protochordate genes and has compared the genetic backgrounds among chordate nutrition processing and alimentary canal/accessory organ systems based on genomic information. Gene expression analyses have revealed spatiotemporal gene expression profiles in protochordate alimentary canals. Additionally, to investigate the basis of morphological diversity in the chordate alimentary canal and accessory organs, evolutionary developmental research has examined developmental transcription factors related to morphogenesis and anterior-posterior pattering of the alimentary canal and accessory organs. In this review, we summarize the current knowledge of molecules involved in nutrition processing and the development of the alimentary canal and accessory organs with innate immune and endocrine roles in protochordates and we explore the molecular basis for understanding the evolution of the chordate digestive system.

Shared hemocyte- and intestine-dominant expression profiles of intelectin genes in ascidian Ciona intestinalis: insight into the evolution of the innate immune system in chordates.

Intelectin is a soluble lectin known as a pattern-recognition receptor for the innate immune system or as an intestinal lactoferrin receptor. Intelectin genes have been identified in a wide range of chordates and the shared expression pattern in their absorptive intestinal regions has been widely recognized. The chordate intelectins have a shared domain structure with a fibrinogen-related domain and an intelectin domain and an additional sequence has been reported only in ascidian Ciona intestinalis intelectins. However, little is known about the molecular features of the ascidian intelectins, including the distribution of the additional sequence in ascidians. Therefore, we focus on the ascidian species that are available for genome DNA sequence searches and survey intelectin genes with special reference to the additional sequence. We also assess the distribution of Ciona intelectin gene transcripts in transparent juveniles and adult specimens by means of in situ hybridization and reveal hemocyte-dominant expressions as well as stomach-exclusive expression. Comparative gene expression analysis with secretory digestive enzymes and absorption-related proteins in Ciona revealed that intelectin and secretory digestive enzymes were expressed in the same region of the stomach epithelium. Since the domain structure of intelectins and the hemocyte-dominant gene expression of intelectins seem relevant to ficolin, intelectin genes may have evolved from a ficolin-like ancestral gene with hemocytic expression in early chordate evolution.

Compartmentalized expression patterns of pancreatic- and gastric-related genes in the alimentary canal of the ascidian Ciona intestinalis: evolutionary insights into the functional regionality of the gastrointestinal tract in Olfactores.

Many heterotrophic animals have a one-way alimentary canal that is essential for their nutrition and sequential steps of the digestive system, namely ingestion, digestion, absorption and elimination, are widely shared among bilaterians. Morphological, functional and molecular knowledge of the alimentary canal has been obtained in particular from mammalian research but the shared features and evolution of these aspects of the highly diverged alimentary canal in the animal kingdom are still unclear. We therefore investigate spatial gene expression patterns of pancreatic- and gastric-related molecules of ascidians (a sister group of vertebrates) with special reference to the functional regionality of the gastrointestinal tract. Genome-wide surveys of ascidian homologs to mammalian exocrine digestive enzyme genes revealed that pancreatic enzymes, namely alpha-amylase, lipase, phospholipase A2, trypsin, chymotrypsin and carboxypeptidase, exist in the ascidian genome. However, an ascidian homolog of the mammalian gastric enzyme pepsin has not been identified, although molecules resembling cathepsin D, a pepsin relative, are indeed present. Spatial expression analyses in the ascidian Ciona intestinalis, by means of whole-mount in situ hybridization, have elucidated that the expression of Ciona homologs of pancreatic- and gastric-related exocrine enzyme genes and of their transcriptional regulator genes is restricted to the Ciona stomach. Furthermore, the expression of these genes is localized to specific regions of the stomach epithelium according to their regionality in the vertebrate digestive system. The compartmentalized expression patterns of Ciona homologs imply primitive and/or ancestral aspects of molecular, functional and morphological bases among Olfactores.

Ordered expression pattern of Hox and ParaHox genes along the alimentary canal in the ascidian juvenile.

The Hox and ParaHox genes of bilateria share a similar expression pattern along the body axis and are known to be associated with anterior-posterior patterning. In vertebrates, the Hox genes are also expressed in presomitic mesoderm and gut endoderm and the ParaHox genes show a restricted expression pattern in the gut-related derivatives. Regional expression patterns in the embryonic central nervous system of the basal chordates amphioxus and ascidian have been reported; however, little is known about their endodermal expression in the alimentary canal. We focus on the Hox and ParaHox genes in the ascidian Ciona intestinalis and investigate the gene expression patterns in the juvenile, which shows morphological regionality in the alimentary canal. Gene expression analyses by using whole-mount in situ hybridization reveal that all Hox genes have a regional expression pattern along the alimentary canal. Expression of Hox1 to Hox4 is restricted to the posterior region of pharyngeal derivatives. Hox5 to Hox13 show an ordered expression pattern correlated with each Hox gene number along the postpharyngeal digestive tract. This expression pattern along the anterior-posterior axis has also been observed in Ciona ParaHox genes. Our observations suggest that ascidian Hox and ParaHox clusters are dispersed; however, the ordered expression patterns along the alimentary canal appear to be conserved among chordates.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

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/adrenomedullin/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.

Gene expression profiles of FABP genes in protochordates, Ciona intestinalis and Branchiostoma belcheri.

Fatty-acid-binding proteins (FABPs) are small intracellular proteins associated with the transportation of fatty acids. Members of the FABPs share similar amino acid sequences and tertiary structures and form, together with a member of the cellular retinol-binding proteins (CRBPs), the intracellular-lipid-binding protein (iLBP) family. In vertebrates, several types of FABP have been isolated and classified into three subfamilies: 2-4. In invertebrates, several FABP-related proteins have been reported in protostomes and amphioxus; however, little is known about the relationship between their phylogenetic positions and expression patterns. We have performed a genome-wide survey of FABP-related genes in protochordates: amphioxus Branchiostoma belcheri and the ascidian Ciona intestinalis. Comprehensive BLAST searches in NCBI and the Ciona Ghost Database by using amino acid sequences of all FABPs have revealed that the ascidian C. intestinalis and amphioxus B. belcheri contain six and seven FABP-related genes in their haploid genomes, respectively. Expression pattern analyses by whole-mount in situ hybridization in Ciona transparent juveniles and serial-section in situ hybridizations in adult amphioxus have revealed that all genes are mainly expressed in the postpharyngeal digestive tract. In particular, the expression of FABP-related genes of subfamily-2 (liver/ileum type) and subfamily-3 (intestinal type) in the ascidian pyloric gland and amphioxus hepatic cecum provides insight into the evolution of hepatic-related structures of chordates and FABP-related genes.

On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates.

As a group closely related to chordates, hemichordate acorn worms are in a key phylogenic position for addressing hypotheses of chordate origins. The stomochord of acorn worms is an anterior outgrowth of the pharynx endoderm into the proboscis. In 1886 Bateson proposed homology of this organ to the chordate notochord, crowning this animal group "hemichordates." Although this proposal has been debated for over a century, the question still remains unresolved. Here we review recent progress related to this question. First, the developmental mode of the stomochord completely differs from that of the notochord. Second, comparison of expression profiles of genes including Brachyury, a key regulator of notochord formation in chordates, does not support the stomochord/notochord homology. Third, FoxE that is expressed in the stomochord-forming region in acorn worm juveniles is expressed in the club-shaped gland and in the endostyle of amphioxus, in the endostyle of ascidians, and in the thyroid gland of vertebrates. Based on these findings, together with the anterior endodermal location of the stomochord, we propose that the stomochord has evolutionary relatedness to chordate organs deriving from the anterior pharynx rather than to the notochord.