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Imine reductase (IRED) from Streptomyces sp. GF3546, one of the first IREDs, was engineered and showed excellent performance at a high concentration of 2-methyl-1-pyrroline and high thermostability at 50 °C.

Sequential regularity control on the n-type conjugated polymers was investigated in this work. The sequentially random polymer produced a near-amorphous structure and a strain-insensitive charge transport performance.

An engineered (

Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

Management of postoperative pain is one of the most important components in postoperative care, because most patients have pain after dental surgery. The aim of this study was to evaluate whether acetaminophen could be an alternative to fentanyl in combination with a nonsteroidal anti-inflammatory drug (NSAID) as an analgesic after dental surgery in cases in which narcotic drugs were contraindicated.Patients were 24- to 54-year-old men who underwent enucleation of a mandibular cyst under general anesthesia. The authors measured time from discontinuation of anesthetic administration until discharge from the operating room and postoperative pain during 4 hours after discharge. They compared these parameters between patients who were intravenously administered an NSAID such as flurbiprofen with fentanyl (NSAID/fentanyl group) and those administered an NSAID with acetaminophen (NSAID/acetaminophen group). Parametric data of time were analyzed using Student t test. Nonparametric data of the analgesic effect were analyzed using Mann-Whitney U test.Time until discharge from the operating room after discontinuation of anesthetics in the NSAID/fentanyl group was significantly longer than that in the NSAID/acetaminophen group (P .05). In contrast, there was no significant difference in analgesic effect between the NSAID/acetaminophen and NSAID/fentanyl groups (P .05).Although recovery time in the operating room of the flurbiprofen and acetaminophen group was markedly shorter than that of the flurbiprofen and fentanyl group, the postoperative analgesic effects of the 2 drugs were equipotent. Therefore, acetaminophen can be an alternative to fentanyl in cases in which narcotic drugs are contraindicated.

Background Joubert syndrome (JS) is a rare autosomal recessive inherited ciliopathy caused by gene mutation. Manifestations can include intermittent dyspnea, apnea, ataxia, and other nervous system abnormalities. Case presentation The patient was a 21-year-old female with JS, severe intellectual disability, cerebral palsy, and epilepsy. Dental caries in both mandibular lower first molars was diagnosed by a local dentist, and the patient was referred to us for further treatment. Although her oral hygiene was good, the dental caries had reached the vicinity of the dental pulp. The caries had developed symmetrically on both first molars and was completely covered with the gingiva. As she was hesitant to receive treatment without anxiolysis, we considered using general anesthesia or intravenous sedation. However, we were recommended against it by the patient's family doctor because of the risk of apneic episodes in JS. Accordingly, dental treatment was performed over multiple days through physical behavior adjustment, while providing oxygenation (3 L/min) in preparation for an apneic episode. The dental treatment was successfully completed with a good postoperative outcome. Conclusions Dental treatment can be safely performed in patients with JS through physical behavior adjustment, thereby minimizing the risk of an apnea attack.

For the improvement of nuclease resistance, four kinds of new modifications through a carbamoylethyl linker were designed. Among them, the 2′-O-[2-N-{2-(benzimidazol-1-yl)ethyl}carbamoylethyl] modification showed 20-fold longer half-life when treated with a 3′ to 5′ exonuclease compared to the 2′-O-methoxyethyl (MOE) modification, which is used in approved drugs. In addition, this large modification did not disturb the binding affinity or RNase H-dependent antisense activity. From these findings, it could be concluded that an adequate linker, such as carbamoylethyl in this study, could extend the utility of 2′-O-modification without loss of the properties of nucleic acids. This strategy would be useful for the development of nucleic acid therapeutics.

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

Background: Chondroitin sulfate (CS) is a sulfated polysaccharide chain that binds to various core proteins to form proteoglycans. The amount and position of sulfate groups in CS are variable among different tissues, and are determined by specific sulfotransferases. Although the ascidians are the closest relatives of vertebrates, the functions of their sulfotransferases have not been studied. Results: The genome of the ascidian Ciona intestinalis contains eight genes encoding proteins similar to chondroitin 6-O-sulfotransferases (C6STs), which appear to have independently diverged in the ascidian lineage during evolution. Among them, Ci-C6ST-like1 and Ci-C6ST-like7 were predominantly expressed in the developing notochord. In addition, they were weakly expressed in the neural tube. The disruption of either one of them affected the convergent extension movement of notochordal cells. Presumptive notochord cells coming from both sides of the embryo did not intercalate. The results suggest that both of them are necessary. In some cases, the anterior neural tube failed to close. Forced expression of Ci-C6ST-like1 or Ci-C6ST-like7 in the notochord restored the normal intercalation of notochordal cells, indicating that the effects of morpholino oligos are specific. Conclusions: Ci-C6ST-like1 and Ci-C6ST-like7 are required for the morphogenesis of the notochord in the ascidian embryo. Developmental Dynamics 243:1637–1645, 2014. © 2014 Wiley Periodicals, Inc.

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

The ascidian Ciona intestinalis is an important model animal for studying developmental mechanisms for constructing the chordate body. Although molecular and embryological techniques for manipulating Ciona genes were developed a long time ago, recent achievements of genome editing in this animal have innovated functional analyses of genes in Ciona. Particularly, knockout of genes in the G0 generation coupled with tissue-specific expression of TALENs enables us to rapidly address gene functions that were difficult using previous methods.

The pharynx, possessing gill slits and the endostyle, is a characteristic of chordates that is a complex of multiple tissues well organized along the anterior-posterior (AP) axis. Although Hox genes show AP coordinated expression in the pharyngeal endoderm, tissue specific roles of these factors for establishing the regional identities within this tissue is largely unknown. Here, we show that Hox1 is essential for the establishment of AP axial identity of the endostyle, a major structure of the pharyngeal endoderm, in the ascidian Ciona intestinalis. We found that Hox1 knockout causes posterior to anterior transformation of the endostyle identity, and Hox1 represses Otx expression and anterior identity, and vice versa. Furthermore, alteration of the regional identity of the endostyle disrupts the formation of body wall muscles, suggesting that the endodermal axial identity is essential for the coordinated pharyngeal development. Our results reveal an essential role of Hox genes for establishment of the AP regional identity in the pharyngeal endoderm and crosstalk between endoderm and mesoderm for the development of chordate pharynx.

Background: Gonadotropin-releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non-reproductive roles. Results: We showed that GnRHs in Ciona are involved in the animal 0 s metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH-3 and tGnRH-5 (the “t” indicates “tunicate”) inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption. Conclusions: This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non-reproductive GnRHs. Developmental Dynamics 00:000–000, 2014. V C 2014 Wiley Periodicals, Inc.

Summary: Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis. genesis 52:431–439, 2014. © 2014 Wiley Periodicals, Inc.

The thermal conductivities of ThO 2 , NpO 2 , (Th, U)O 2 , (Th, Pu)O 2 and (U, Np)O 2 have been investigated by molecular dynamics (MD) simulation up to 2000 K using the Busing–Ida potential function with partial ionic charges. In the present study, the thermal conductivity was calculated mainly by the Green–Kubo formula in the equilibrium MD scheme. The thermal conductivities of above actinide dioxides decreased with the increase of temperature due to the phonon–phonon interaction (Umklapp process). Concerning the composition of solid solutions, the decrease in thermal conductivity of (Th, Pu)O 2 is great as compared to other ones. Various MD calculations elucidated that this result was caused by phonon scattering by lattice defects as additives rather than the phonon–phonon interaction, and that the lattice strain dominantly contributed to it.

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.

A case of palmoplantar pustulosis and hyperthyroidism following orthognathic surgery is presented. Both diseases may have been related to allergic phenomena.

The oxygen potential of hypo-stoichiometric (U0.8La0.2)O2−x was measured as a function of oxygen-to-metal ratio (2 − x) in the temperature range from 1173 K to 1473 K. Compared with uranium oxides doped with other lanthanides, e.g. Lu, Er, Gd and Nd, the relationship between Δ G ¯ O 2 and the oxygen-to-metal ratio obtained for this sample showed almost the same trend, and for the relatively large oxygen non-stoichiometry x, its significance was comparable with that of (U0.8Gd0.2)O2−x. However, the detailed variation of Δ H ¯ O 2 and Δ S ¯ O 2 was different among these uranium oxides, especially in the near O/M = 2.0 region. Furthermore, the oxygen defect structure was evaluated from the relationship between log P O 2 and log x, and Δ G ¯ O 2 value of lanthanide-doped uranium oxide was discussed in terms of lanthanide ionic radius.

Nodal signaling plays an essential role in the establishment of left–right asymmetry in various animals. However, it is largely unknown how Nodal signaling is involved in the establishment of the left–right asymmetric morphology. In this study, the role of Nodal signaling in the left–right asymmetric ocellus formation in the ascidian, Ciona intestinalis was dealt with. During the development of C. intestinalis , the ocellus pigment cell forms on the midline and moves to the right side of the midline. Then, the photoreceptor cells form on the right side of the sensory vesicle (SV). Ci-Nodal is expressed on the left side of the SV in the developing tail bud embryo. When Nodal signaling is inhibited, the ocellus pigment cell form but remain on the midline, and expression of marker genes of the ocellus photoreceptor cells is ectopically detected on the left side as well as on the right side of the SV in the larva. Furthermore, Ci-Rx , which is essential for the ocellus differentiation, turns out to be negatively regulated by the Nodal signaling on the left side of the SV, even though it is required for the right-sided photoreceptor formation. These results indicate that Nodal signaling controls the left–right asymmetric ocellus formation in the development of C. intestinalis .

Pt(II) and Pt(IV) porphyrin pincer complexes were synthesized and characterized. Their structures and electronic properties were influenced by the valence state of platinum at the outer coordination site. The considerable distortion in the porphyrin core of the Pt(II) complex was relieved in the Pt(IV) complex, due to the change of the coordination geometry of platinum. Oxidation of the phenylplatinum(II) pincer complex with iodine induced a facile carbon−carbon bond formation reaction at the meso position via reductive elimination.

Left-right asymmetric expression of Pitx as well as of Nodal has been observed in some ascidian species, but a mechanism that regulates the asymmetric expression of Pitx remains largely unclear. We addressed the transcription regulatory mechanism of the left-right asymmetric expression of Pitx gene in the ascidian, Ciona intestinalis. We first identified an intronic enhancer that drives Ci-Pitx left-sided expression in the epidermis and found that a single FoxH1 binding site present in this enhancer is essential for its activity. Also, we have shown that the enhancer requires Nodal signaling to drive the Ci-Pitx expression in the left epidermis. In C. intestinalis, left-sided expression of the Nodal gene has not been reported so far. We have confirmed that Ci-Nodal is expressed in the left epidermis of embryos at the tailbud stage only when they are allowed to develop within the chorion. Then, to test the importance of the FoxH1 binding site, we carried out knock down experiments using morpholino antisense oligonucleotides against Ci-FoxHa, the only FoxH gene that is expressed in the same stage as the left-sided expression of Ci-Pitx is observed. Knocking down of the function of Ci-FoxHa led to the down regulation of the expression of Ci-Pitx in the left epidermis. The present results suggest that the regulatory mechanism controlling the left-right asymmetric expression of the Pitx genes is well conserved between C. intestinalis and vertebrates.

Gonadotropin-releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non-reproductive roles.We showed that GnRHs in Ciona are involved in the animal's metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH-3 and tGnRH-5 (the "t" indicates "tunicate") inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption.This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non-reproductive GnRHs.

Chondroitin sulfate (CS) is a sulfated polysaccharide chain that binds to various core proteins to form proteoglycans. The amount and position of sulfate groups in CS are variable among different tissues, and are determined by specific sulfotransferases. Although the ascidians are the closest relatives of vertebrates, the functions of their sulfotransferases have not been studied.The genome of the ascidian Ciona intestinalis contains eight genes encoding proteins similar to chondroitin 6-O-sulfotransferases (C6STs), which appear to have independently diverged in the ascidian lineage during evolution. Among them, Ci-C6ST-like1 and Ci-C6ST-like7 were predominantly expressed in the developing notochord. In addition, they were weakly expressed in the neural tube. The disruption of either one of them affected the convergent extension movement of notochordal cells. Presumptive notochord cells coming from both sides of the embryo did not intercalate. The results suggest that both of them are necessary. In some cases, the anterior neural tube failed to close. Forced expression of Ci-C6ST-like1 or Ci-C6ST-like7 in the notochord restored the normal intercalation of notochordal cells, indicating that the effects of morpholino oligos are specific.Ci-C6ST-like1 and Ci-C6ST-like7 are required for the morphogenesis of the notochord in the ascidian embryo.

Infertility caused by ovarian or tubal problems can be treated using In Vitro Fertilization and Embryo Transfer (IVF-ET); however, this is not possible for women with uterine loss and malformations that require uterine reconstruction for the treatment of their infertility. In this study, we are the first to report the usefulness of decellularized matrices as a scaffold for uterine reconstruction. Uterine tissues were extracted from Sprague Dawley (SD) rats and decellularized using either sodium dodecyl sulfate (SDS) or high hydrostatic pressure (HHP) at optimized conditions. Histological staining and quantitative analysis showed that both SDS and HHP methods effectively removed cells from the tissues with, specifically, a significant reduction of DNA contents for HHP constructs. HHP constructs highly retained the collagen content, the main component of extracellular matrices in uterine tissue, compared to SDS constructs and had similar content levels of collagen to the native tissue. The mechanical strength of the HHP constructs was similar to that of the native tissue, while that of the SDS constructs was significantly elevated. Transmission electron microscopy (TEM) revealed no apparent denaturation of collagen fibers in the HHP constructs compared to the SDS constructs. Transplantation of the decellularized tissues into rat uteri revealed the successful regeneration of the uterine tissues with a 3-layer structure 30 days after the transplantation. Moreover, a lot of epithelial gland tissue and Ki67 positive cells were detected. Immunohistochemical analyses showed that the regenerated tissues have a normal response to ovarian hormone for pregnancy. The subsequent pregnancy test after 30 days transplantation revealed successful pregnancy for both the SDS and HHP groups. These findings indicate that the decellularized matrix from the uterine tissue can be a potential scaffold for uterine regeneration.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event.

Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.

The selective N-methyl-d-aspartate (NMDA) channel blocker MK-801 is known to induce no loss of the righting reflex (LORR) and to stimulate catecholaminergic (CAergic) neurons in rodents, playing a crucial role in arousal. We examined whether MK-801 in combination with CA receptor ligands, which inhibit CAergic neuronal activities, could induce anesthesia including LORR. All drugs were administered systemically to mice. To assess anesthesia, three different behaviors were used: loss of nociceptive response (analgesia in the free-moving state without LORR), LORR, and loss of movement in response to noxious stimulation (immobility under LORR). A very large dose of MK-801 (50 mg/kg) induced neither analgesia nor LORR. In contrast, MK-801 in combination with a small dose of the dopamine (DA) receptor antagonist haloperidol (0.2 mg/kg) dose-dependently produced LORR with a 50 % effective dose (ED50) of 1.6 (0.9–3.0; 95 % confidence limit) mg/kg, but not immobility. The α2-adrenoceptor agonist dexmedetomidine induced not only analgesia, but also immobility in animals treated with MK-801 (5 mg/kg) plus haloperidol (0.2 mg/kg), which then lost their righting reflex. The ED50 value of 0.26 (0.10–0.66) mg/kg (various doses of dexmedetomidine plus a fixed dose of MK-801 and haloperidol) for immobility was approximately three-fold larger than that of 0.09 (0.03–0.23) mg/kg (dexmedetomidine plus vehicle saline) for analgesia. This may occur, as LORR induced by MK-801 plus haloperidol inhibits the pain suppression system. The other ligands had little or no effect. The DAergic stimulant actions of MK-801 may mask its LORR effects by NMDA channel blockade.

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr-Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr-Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene as an enhancer region that drives the transcription of Hr-Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr-Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr-Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates.