Tachykinin and Tachykinin Receptor of an Ascidian, Ciona intestinalis

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.

In protostomes, two types of TK-like peptides, invertebrate TK and TK-related peptides (TKRPs), have so far been identified. Peptides of the former group, containing the identical C-terminal TK consensus motif, are expressed exclusively in the salivary gland and are devoid of any activity on the cognate tissues, indicating that the TK-like peptides are not functional counterparts of vertebrate TKs (9,19,20). TKRPs exert a TK-like contractile activity, and the expression of the TKRP gene is observed in the central nervous system (20). However, they contain the analogous -FX 1 (G/A)X 2 R-NH 2 consensus, and TKRP precursors encode multiple TKRP sequences (20 -23), which are totally distinct from those of vertebrate TKs. In addition, no TKRPs have ever been isolated from vertebrates. In earlier studies, SP-and/or NKA-like immunoreactivities were detected in the central nervous system and several peripheral tissues of ascidians by immunohistochemical analyses and radioimmunoassays (24 -28). However, neither molecular nor functional characteristics of authentic ascidian TKs and their receptor have ever been elucidated, and no reproducible findings have been provided by previous immunohistochemical studies (24 -28). Since investigation of TKs or TKRPs in proto-chordates is expected to provide crucial findings concerning not only the biological roles of TKs or TKRPs in protochordates but also the evolutionary origins of the structures and functions of the TK family, we explored TK peptides and its receptor in an ascidian, Ciona intestinalis, which belongs to protochordates as a basal chordate, namely an emerging deuterostome model animal (29 -31). In this work, we present the structure, localization, and reactivity of Ciona TK, Ci-TK, and its receptor, Ci-TK-R, suggesting biological roles of the TK family in protochordates and the features of Ci-TK and Ci-TK-R as prototypes of vertebrate TK peptides and receptors.

EXPERIMENTAL PROCEDURES
Animals-Adults of C. intestinalis were cultivated and collected at the Maizuru Fisheries Research Station of Kyoto University, and maintained in sea water at 18°C.
PCR Primers-All sequences of PCR primers are summarized in Supplemental Table I.
Identification of the Ci-TK and Ci-TK-R cDNAs-Total RNA (0.5 g) from the neural complex was reverse-transcribed to the template cDNA at 55°C for 60 min using the oligo(dT) anchor primer and the avian myeloblastosis virus reverse transcriptase supplied in the 5Ј/3Ј-rapid amplification of the cDNA ends (RACE) kit (Roche Applied Science). The partial Ci-TK cDNA was obtained by PCR using the primers identical to nucleotides (nt) 46 -66 and complementary to nt 499 -518. RACE was performed using the gene-specific primers complementary to nt 268 -288 (for 5Ј-RACE) and nucleotides identical with nt 268 -288 (for 3Ј-RACE), respectively. Similarly, the Ci-TK-R cDNA was obtained by RT-PCR using the primers identical to nt 20 -40 and complementary to nt 1464 -1485 followed by nested PCR with primers identical to nt 45-68 and complementary to nt 1391-1410. 5Ј-RACE with primers complementary to 350 -371 and 379 -398, and 3Ј-RACE with primers identical to nt 1261-1280 and nt 1310 -1329 were subsequently performed. Subcloned inserts were sequenced on an ABI PRISM TM 310 Genetic Analyzer (Applied Biosystems) using a Big-Dye sequencing kit (Applied Biosystems) and universal primers (SP6 and T7 primers).
Mass Spectrometry (MS)-Ten Ciona neural complexes were pulverized by grinding under liquid nitrogen and extracted in 20 ml of boiled water. The resultant extract was eluted using a Sep-pak plus C-18 cartridge (Waters; Tokyo, Japan), and the eluate was evaporated and lyophilized. To acquire MS/MS spectra of Ci-TKs, the crude peptide was dissolved in 50% (v/v) methanol containing 0.1% formic acid, followed by observation of the spectra for Ci-TK-I and -II with a Q-TOF tandem mass spectrometer equipped with a Z-spray nanoelectrospray interface (Micromass, Manchester, UK). The needle voltage was optimized at 1000 V; the cone voltage was set at 50 V. Argon was used as the collision gas, and the collision gas energy was set at 28 V.

Functional Analysis of Ci-TK-R Expressed in Xenopus
Oocytes-The open reading frame region of Ci-TK-R cDNA was amplified and inserted into the Xenopus expression vector pSPUTK (Stratagene). The cRNA was prepared from the plasmid linearized with HpaI using SP6 RNA polymerase (Ambion, Austin, TX). 50 nl of the cRNA solution (0.05 g/l) were injected into oocytes. The oocytes were incubated for 2-4 days at 17°C and transferred to ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES (pH 7.6)). The oocytes were voltage-clamped at Ϫ80 mV. The dose-response data and the EC 50 values of the experiment were analyzed using Origin 6.1 software (Microcal Software, Tokyo). For the bioassay, the guinea pig ileum was a gift from Dr. Toshiaki Fujii. The contractile effects of Ci-TKs and other TKs were observed as described previously (19).
Southern Hybridization of RT-PCR Products-The primer sets used for Ci-TK cDNA were identical with nt 20 -40 in the Ci-TK cDNA and complementary to nt 582-602; the primer sets used for Ci-TK-R cDNA were identical with nt 45-68 in the Ci-TK-R cDNA and complementary to nt 752-773, and the primer sets used for Ciona ␤-actin cDNA were identical with nt 254 -274 in the Ciona ␤-actin cDNA and complementary to nt 845-864. PCR was performed for 25 cycles for amplification of the Ci-TK cDNA and for 35 cycles for the Ci-TK-R cDNA consisting of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C. PCR products were resolved on a 1.5% agarose gel followed by transfer to a Hybond N ϩ membrane (Amersham Biosciences). Preparation of digoxigenin (DIG)labeled cDNA probes, hybridization, and detection were performed in accordance with the DIG system protocol (Roche Applied Science).
In Situ Hybridization-The Ci-TK cDNA fragment (nt 47-558) was inserted into the PST 18 vector (Roche Applied Science), and the linearized plasmid was supplied to the synthesis of DIG-labeled Ci-TK RNA probe using a DIG RNA labeling kit (Roche Applied Science) Whole-mount in situ hybridization of the juvenile and adult neural complex and endostyle were performed as previously described (32,33). The Ciona digestive tracts were dissected and fixed in Bouin's fluid at 4°C overnight. Preparation of 5-m serial sections, hybridization, washing, and detection were carried out as previously reported (34). No positive signals were observed when sense probes were used, confirming the specificity of hybridization.

Identification of the Ci-TK Peptides, cDNA, and Genomic
Structure-In an attempt to detect a Ciona TK cDNA, we performed BLAST searches on the draft genome data base of C. intestinalis (available on the World Wide Web at ghost-.zool.kyoto-u.ac.jp/indexr1.html) (35). Application of only ␥-PPTA to the data base searches resulted in the detection of several EST clones (Cluster ID CLSTR36631 on the Ciona genome data base). These clones were found to encode an identical open reading frame including two putative TK sequences with the FXGLM vertebrate TK consensus motif. This was reminiscent of the structural organization of ␥-PPTA (7,12,13). The full-length cDNA was cloned from the neural complex by RT-PCR followed by 5Ј-and 3Ј-RACE. As shown in Fig. 1A, the deduced amino acid sequence encoded two putative TK sequences flanked by a Gly C-terminal amidation signal at their C termini and typical endoproteolytic sites at both termini, suggesting that two TK peptides, HVRHFYGLM-NH 2 and ASFTGLM-NH 2 , are produced from the precursor. Thus, we designated these two peptides as Ci-TK-I and Ci-TK-II, respectively. The in silico analyses of the Ci-TK genomic structure using the JGI Ciona genome project data base (available on the World Wide Web at www.jgi.doe.gov/programs/ciona. html) verified that the Ci-TK gene consists of six exons. Notably, both Ci-TK-I and -II sequences were encoded in the third exon ( Fig. 1B), contrary to SP and NKA, which are encoded in the fifth and seventh exon of the vertebrate PPTA gene, respectively (7,12,13).
To investigate the presence of Ci-TK peptides as mature forms, we performed Q-TOF MS/MS analysis of the peptide fraction of the ascidian neural complex. Fig. 2 demonstrates the fragmentation patterns, each of which was compatible with HVRHFYGLM-NH 2 and ASFTGLM-NH 2 , respectively. In ad- dition, no mass values for putative N-terminally extended forms of Ci-TK-I or II were observed. In combination, these data confirmed that both Ci-TK-I and II are yielded from the precursor. Consequently, we concluded that TKs are conserved in protochordates as well as vertebrates.
Sequence Comparison of Ci-TKs with Other TKs-Ci-TK-I was found to possess a Tyr and an Arg at positions 4 and 7 from the C terminus, respectively (Table I), which is in good agreement with the fact that TKs with an aromatic amino acid and a neutral or basic amino acid at each of these positions show the binding selectivity to NK1, the SP-selective TK receptor (1,7). On the other hand, Ci-TK-I lacks Pro, which is seen at position 8 and/or 10 from the C terminus of SP and several submammalian SP-like TKs (1,7). In addition, the N-terminal region of Ci-TK-I (HVRH) displayed no significant similarity to any other TKs. Ci-TK-II shared a Ser with NKA and several amphibian and fish TKs (36 -39) at position 6 from the C terminus as shown in Table I. However, the most outstanding feature of Ci-TK-II is that this ascidian TK is composed of only 7 amino acids, whereas other known TKs include 9 -11 amino acids (1,7). Moreover, the Thr at position 4 from the C terminus of Ci-TK-II is regarded as a unique amino acid residue, given that SP and its related TKs contain an aromatic amino acid, whereas an aliphatic amino acid is located at this position in NKA, NKB, and their counterparts (1,7). Therefore, these indicated the distinct sequence property of Ci-TK-II.
Molecular Characterization of Ci-TK-R-The Ciona genomic data base search with mammalian TK and protostome TKRP receptors provided a GPCR-like protein (Cluster ID 15345), which displayed the highest sequence similarity to mammalian NK2 receptor. The full-length sequence of the Ciona putative GPCR was determined by combination of RT-PCR, 3Ј-RACE, and 5Ј-RACE. The deduced amino acid sequence was shown to harbor the seven hydrophobic transmembrane regions and several sites that are conserved in TK receptors (Fig. 3). A sequence comparison verified that the transmembrane domain of the putative Ciona GPCR displayed high amino acid sequence similarity (30 -43%) to those of mammalian TK receptors and protostome TKRP receptors (Fig. 3). Taken together, these results allowed us to presume that the putative Ciona GPCR is a candidate for a Ci-TK receptor, Ci-TK-R.
The response and binding selectivity of TK receptors to their agonists were evaluated by functional expression analysis using Xenopus oocytes (1, 7, 15-18, 20, 40). Thus, we examined whether the Ci-TK-R expressed in Xenopus oocytes was activated by Ci-TKs. Each Ci-TK was added to a Ci-TK-R-expressing oocyte every 20 min at the indicated concentrations in order to prevent desensitization of the receptor. As shown in Fig. 4A, application of Ci-TK I to the Ci-TK-R-expressing Xenopus oocytes evoked a typical calcium-dependent inward chloride current. A maximal response was observed at more than 20 nM, and the EC 50 was calculated to be ϳ6.92 nM by a dose-response curve of current shift (Fig. 4B). These results revealed that Ci-TK-I is undoubtedly an endogenous ligand of Ci-TK-R. This ascidian TK receptor also showed comparable responses to SP and NKA (Fig. 4B), indicating that Ci-TK-R lacked the binding selectivity typical for NK1 to -3 (1, 2, 7). It is noteworthy that this is the first report on the TK receptor that was shown to be equivalently activated by different TK peptides. However, another endogenous TK, Ci-TK-II, was shown to exhibit ϳ100fold less potent activity (EC 50 ϭ 641 nM) than Ci-TK-I (Fig. 4B), suggesting the ligand selectivity of Ci-TK-R to Ci-TK-I. Such reactivity was also observed by the functional analysis of the Ci-TK-R expressed in HEK 293 cells (results not shown), excluding the possibility that this phenomenon resulted from the heterologous expression. Moreover, co-application of Ci-TK-I and Ci-TK-II showed no reduction of the activation of Ci-TK-R by Ci-TK-I (not shown), confirming that Ci-TK-II has no significant antagonistic activity against Ci-TK-R. Altogether, these results led to the conclusion that the TK family is functional in protochordates. In addition, homology data base searches detected only Ci-TK-R as a TK receptor-related GPCR, and the second and third most homologous GPCRs (Cluster ID 35041 and 02264 on the Ciona genome data base), which display the highest sequence similarity to Carassius somatostatin receptor III and mouse orexin receptor II, respectively, were not responsive to any Ci-TKs in the same functional analysis (data not shown). These suggested that Ci-TK-R was a single GPCR for Ci-TKs, although Ci-TK-R possesses the ligand selectivity to Ci-TK-I.
Contractile Activities of Ci-TKs on Gut Tissues-TKs are known to induce a characteristic contraction of gut tissues (1,7,19,20). Nevertheless, Ci-TKs had no contractile effect on the Ciona gut (data not shown), most likely due to the absence of intestinal muscular tissues in the ascidian gut (29). Instead, Ci-TK-I was found to elicit a typical contraction of the guinea pig ileum, as seen in administration of SP (Fig. 5). Ci-TK-II was much less potent than Ci-TK-I. The markedly reduced contractile activity of Ci-TK-II is consistent with the faint stimulation of Ci-TK-II on Ci-TK-R expressed in Xenopus oocytes at physiological concentrations (Fig. 4B). In combination, these results show that Ci-TKs are conferred with the essential pharmacological property of the TK family peptides.
Expression of the Ci-TK Gene and Ci-TK-R Gene-Southern blotting analysis of the RT-PCR products showed that the expression of the Ci-TK gene was present predominantly in the neural complex and endostyle and in the digestive system at low levels, whereas the Ci-TK-R mRNA was localized in the neural complex, digestive system, and gonad to a similar degree and less in the endostyle (Fig. 6). These results suggest the central and peripheral function of Ci-TKs in these tissues for feeding and sexual behavior as not only brain/gut peptides but also endocrine/paracrine molecules in the endostyle. To further examine the expression profile of Ci-TK gene, we performed in situ hybridization of the embryos, larvae, juveniles, and the adult neural complex, endostyle, and intestine. Whole-mount in situ hybridization of the embryos at each stage and larvae detected no signals (data not shown). In the juveniles, an intense signal was observed in the brain ganglion (Fig. 7, A and  B). Furthermore, the abundant expression of the Ci-TK gene was detected in neurons situated in both the cortex and medulla regions of the adult brain ganglion (Fig. 7C). In the endostyle, the Ci-TK mRNA was present exclusively in terminal cells of zone 7 proximal to zone 8 (Fig. 7, D and E), which is believed to be a homologous component of the vertebrate thyroid gland (29). Interestingly, the Ci-TK-expressing regions were shown to contain numerous secretory granules (29), suggesting that Ci-TKs act as an endocrine/paracrine molecule in the endostyle. Localization of the Ci-TK mRNA was also seen in unidentified small cells scattered on the intestinal epithelial layer (Fig. 7F). Taken together, the localization of the Ci-TK and Ci-TK-R transcripts suggest not only multiple functionality of Ci-TKs in the adult ascidian rather than the developmental stages and larvae but also the functional conservation between Ci-TKs and vertebrate TK as a brain/gut peptide and an endocrine/paracrine substance. DISCUSSION Ascidians have been recognized as an excellent model organism for developmental and evolutionary studies due to their simple body plan and their critical evolutionary position as basal chordates (29 -31). Recently, in addition to these advantages, the genome data base for C. intestinalis was constructed (35), and morpholino DNA gene silencing (41) and transgenic technology (42) for the ascidian have emerged, leading to expansion of the applicability of ascidians to other fields, including neuroscience and endocrinology, as the only invertebrate chordate model, which is more suitable than protostome models such as Drosophila and C. elegans in regard to the phylogenetic distance. However, only two peptides, GnRH and cionin (CCK/gastrin-related peptide), have so far been identified from ascidians (43)(44)(45), although the presence of TK-like compounds in ascidians was implied by previous immunohistochemical analyses and radioimmunoassays for several ascidian tissues (24 -28). Furthermore, TKRPs, but not TKs, have been found in invertebrates, which complicates the evolutionary aspect of the TK family (7,20). In this study, we showed the structure, activity, and localization of a TK peptide, Ci-TK, and its receptor, Ci-TK-R, of an ascidian, C. intestinalis. This is the first characterization of an authentic TK peptide and its receptor from invertebrates as well as protochordates, namely nonvertebrate species. Ci-TK contains the vertebrate TK consensus motif, and the structural organization of prepro-Ci-TK displays a resemblance to those of vertebrate ␥-PPTA encoding SP and NKA (Fig. 1). Moreover, Ci-TKs elicited a typical TK-like contraction of the guinea pig ileum (Fig. 5). These results provide indisputable evidence that the TK family, not TKRPs, is evolutionarily conserved in protochordates. Nevertheless, obvious differences between the Ci-TK gene and vertebrate PPTA genes were also found; two Ci-TK sequences are encoded in the same exon (Fig. 1B), suggesting no alternative production of Ci-TK-I and -II. In contrast, vertebrate SP and NKA sequences are located in separate exons in the PPTA gene, which enables generation of splicing variants encoding SP alone or both SP and NKA (12,13). In addition, we have detected neither additional TK peptides nor TK genes. These findings indicate that the "prototype" TK gene, organized similarly to the Ci-TK gene, originally encoded two TKs in the same exon and then was divided by intron inserts followed by acquisition of the alternative splicing system during the divergence of the ancestral gene into PPTA, PPTB, and PPTC genes in the evolutionary process of vertebrates.
Ci-TK-I was found to contain a Tyr and an Arg at position 4 and 7, respectively, from the C terminus (Table I). These are consistent with the finding that an aromatic amino acid and a basic or neutral amino acid is located at each of these positions in SP and its submammalian counterparts (1, 7). Accordingly, the presence of these amino acids in the Ci-TK-I sequence is compatible with the potent activity on the Ci-TK-R (Fig. 4) and the guinea pig gut (Fig. 5). On the other hand, Ci-TK-II elicited 100-fold less potently the receptor activation and contractile activity than did Ci-TK-I (Figs. 4 and 5), although the Ser at position 6 from the C terminus is conserved among Ci-TK-II, NKA, and several fish and amphibian TKs (Table I). Such low activities of Ci-TK-II can be interpreted in three ways. First, the Ci-TK-II sequence is shorter (7 amino acids) than any other TKs (more than 9 amino acids), including Ci-TK-I. If the short sequence is correlated with the low activities (Figs. 4 and 5), the binding selectivity of Ci-TK-R is highly likely to be simply length-dependent rather than sequence-dependent, since Ci-TK-R is equivalently activated by Ci-TK-I, SP, and NKA (Fig.  4B). Second, the low activity of Ci-TK-II may be attributed to the Thr residue at position 4 from the C terminus. The location of a Thr at this position has not been found in any vertebrate TKs; it is well established that SP-like and NKA/B-like TKs harbor an aromatic amino acid and an aliphatic amino acid at The SP-typical amino acids (an aromatic residue at position 4 and a basic/neutral residue at position 7 from the C terminus) in the Ci-TK-I sequence are underlined. Ci-TK-II possesses a Thr at position 4 from the C terminus instead of an aromatic residue typical of SP or an aliphatic one typical of NKA, although the Ser (underlined) at position 6 from the C terminus is conserved among Ci-TK-II, NKA, and several submammalian TKs. the corresponding position (1,7), and these amino acid residues play a crucial role in the binding selectivity to NK1 to -3 (1, 7). Thus, these findings imply the correlation of the Thr with the low activities of Ci-TK-II. Alternatively, the possibility cannot be absolutely ruled out that Ciona GPCRs other than Ci-TK-R or non-GPCR proteins might be an authentic receptor for Ci-TK-II, although no additional Ciona GPCR homologous to TK or TKRP receptors was detected by our data base searching. To address these issues, an investigation of the relationship between sequence/structure and activity on Ci-TK-II is under way. Ci-TK-I, SP, and NKA exerted the equivalent activity on Ci-TK-R (Fig. 4B). Furthermore, the Ciona genome data base searching detected only Ci-TK-R as a GPCR with high sequence similarity to TK or TKRP receptors. In combination, these results indicate that the binding selectivity of Ci-TK-R to Ci-TKs is distinct from the sequence-dependent selectivity of NK1-3. Consequently, it is suggested that the ancestral TK receptor very likely possesses no significant ligand selectivity and that the ligand selectivity of TK receptors, along with the alternative production of TK ligands, were established during generation of NK1-3 in vertebrates, which is also supported by the phylogenetic analysis showing that NK1-3 originated from a common ancestral gene in the early process of vertebrate evolution (11,46). In keeping with this, the biological significance of such binding selectivity of Ci-TK-R to Ci-TKs is raised as a new question in light of the simultaneous production of Ci-TKs and the existence of the Ci-TK-R as the sole TK receptor in the ascidian, although Ci-TK-II may be a nonfunctional ligand due to its markedly low activity.
The tissue distribution of ascidian TK-like peptides were investigated using antibodies against SP or NKA, which led to different findings mainly due to the low specific reactivity of the antibodies (24 -28). In this study, we have clearly localized the expression of the Ci-TK and Ci-TK-R genes by RT-PCR and in situ hybridization. The Ci-TK gene was expressed in the adult neural complex, intestine, and endostyle (Fig. 6), and the expression of the Ci-TK gene was initially found in the neural complex of the juvenile (Fig. 7, A and B). Furthermore, the Ci-TK-R transcript was distributed in the neural complex, intestine, endostyle, and gonad of adults (Fig. 6). These results indicate that Ci-TK plays a crucial role in the essential behaviors of the adult ascidian, such as feeding and sexual behavior, and that such biological functions of TK might have been es-  The expression of the Ci-TK gene in neurons of both the cortex and the medulla regions in the brain ganglion (Fig. 7C) suggests the multiple roles of Ci-TK in neural communication and central regulation of peripheral tissues. In particular, it is strongly suggested that gonad functions are subjected to direct regulation by cerebral Ci-TK, given that the Ci-TK-R gene was abundantly expressed in the gonad despite the absence of the Ci-TK mRNA in this tissue (Fig. 6). In recent studies, ␤and ␥-PPTA were shown to be localized in Leydig cells of the human and mouse testis (47), and the expression of all PPTA, PPTB, and NK1 to -3 genes was also observed in nonneuronal cells of the mammalian uterus (48,49). Indeed, an elevation of some sexual steroid hormone-like substances was observed upon administration of Ci-TK-I on the ascidian gonad. 2 A more precise mechanism for the biological effects of Ci-TK-I on the gonad is now being examined.
The striking feature is that the Ci-TK gene is expressed in terminal cells residing in the dorsal terminus of zone 7 in the endostyle (Fig. 7, D and E). The endostyle is a pharyngeal organ that is responsible for secretion of mucus proteins for internal filter feeding and uptake of iodine (29). Furthermore, thyroid peroxidase activity and the peroxidase gene expression were detected specifically in zone 7 (50). Consequently, the endostyle is believed to be a functional antecedent of the vertebrate thyroid gland (29,50). The Ci-TK-expressing region was found to contain numerous secretory vesicles (29), which is indicative of the function of this region as a secretory gland. Combined with these findings, the specific expression of the Ci-TK gene in the terminal cells strongly suggests some endocrine/paracrine roles of Ci-TKs released from this region in the control of thyroid-like functions the endostyle. In mammals, no biological effect of TKs on the thyroid gland has ever been elucidated (51)(52)(53), although immunoreactivity against SP was observed in the nerve fibers (52). However, hypothyroidism by removal of the thyroid gland induced an increase in SP and NKA peptides and up-regulation of ␥-PPTA mRNA in the thyrotroph of the anterior pituitary (51), whereas hyperthyroidism caused by administration of excess thyroxin resulted in a decrease of both peptidic and transcriptional TK products (51)(52)(53), indicating the unknown functional correlation between pituitary TKs and the thyroid gland. Taken together, our data lead to a presumption that endocrine/paracrine functions of Ci-TK for the ascidian endostyle evolved into the thyroid glandpituitary TK regulatory system. Therefore, investigation of the biological roles in endostyle Ci-TK is expected to provide a crucial clue to the understanding of an evolutionary process from the endostyle to the thyroid gland. Functional studies of Ci-TK-I on the endostyle are currently in progress.
Also of interest is the biological role of Ci-TK in small cells underlying the intestinal epithelial layers (Fig. 7F). These small cells have yet to be functionally characterized, but immunoreactivities against several human neuropeptides were also detected in such small cells (24,29), suggesting that the intestinal small cells are responsible for production and release of paracrine/endocrine substances including Ci-TK. Mammalian gut TKs (SP and NKA) are produced mainly by intrinsic enteric neurons, and SP and NKA released by enteric neurons participate in muscle contraction, electrolyte and fluid secretion, tissue homeostasis, and afferent sensory function (1, 2, 6, 7). The major role of Ci-TK produced in the intestine, unlike that of mammalian gut SP/NKA, cannot be the contraction of the muscle, given that the ascidian intestine has almost abol-ished contractile action (29). Thus, the expression of the Ci-TK gene in the small cells indicates the possibility that gut Ci-TK is involved in the control of other gut functions as mentioned above and that such functions of TKs in the gut were established in the common ancestral chordate. In summary, we have identified TK and its receptor from a protochordate, C. intestinalis. Our data not only revealed conservation of essential structural organization and neuropeptidic function of the TK family in chordates but also established Ci-TKs and Ci-TK-R as the evolutionary origins of TKs and their receptors.