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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., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ambulacrarians (echinoderms and hemichordates) are a sister group to chordates; thus, their larval cell-types may provide clues about evolution of chordate body plans. Although most genic information accumulated to date pertains to sea urchin embryogenesis, starfish embryogenesis represents a more ancestral mode than that of sea urchins. We performed single-cell RNA-seq analysis of cell-types from gastrulae and bipinnarial larvae of the starfish, Patiria pectinifera, and categorized them into 22 clusters, each of which is composed of cells with specific, shared profiles of development-relevant gene expression. Oral and aboral ectoderm, apical plate, hindgut or archenteron, midgut or intestine, pharynx, endomesoderm, stomodeum, and mesenchyme of the gastrulae, and neurons, ciliary bands, enterocoel and muscle of larvae were characterized by expression profiles of at least two relevant transcription factor genes and signaling molecular genes. Expression of Hox2, Hox7, Hox9/10, and Hox11/13b was detected in cells of clusters that form the larval enterocoel. By comparing homologous gene expression profiles in chordate embryos, we discuss and propose how the chordate body plan evolved from a deuterostome ancestor, from which the echinoderm body plan also evolved., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin is known to promote sodium transport and regulate gluconeogenesis in renal proximal tubules. Although protein kinase B (also known as Akt) and mammalian target of rapamycin complexes (mTORC) have been established as key regulators in the insulin signaling pathway, their roles in proximal tubules are poorly understood. To help define this, we examined the components of insulin signaling in sodium transport and gluconeogenesis in isolated human and rat proximal tubules, and also investigated the role of insulin in sodium handling and mTORC1 in insulin signaling in vivo. In isolated human and rat proximal tubules, Akt and mTORC1/2 inhibition suppressed insulin-stimulated sodium-bicarbonate co-transporter 1 (NBCe1) activity, whereas mTORC1 inhibition had no effect. Akt2 and mTORC2 gene silencing largely inhibited insulin-stimulated NBCe1 activity, whereas silencing of Akt1 and mTORC1 had no effect. Furthermore, insulin decreased sodium excretion, and this effect depended on phosphoinositide 3 kinase in vivo. Moreover, insulin reduced glucose production in rat proximal tubules and the expression of gluconeogenic genes in human and rat proximal tubules. Akt and mTORC1 inhibition largely abolished the observed insulin-mediated inhibitory effects. Gene silencing of insulin receptor substrate 1 (IRS1), Akt2, mTORC1, and mTORC2 also abolished insulin-mediated inhibition of gluconeogenesis. Additionally, in vivo, mTORC1 inhibition abolished insulin-mediated inhibitory effects in rat proximal tubules, although not in liver. These results indicate that insulin-stimulated proximal tubule sodium transport is mediated via the Akt2/mTORC2 pathway, whereas insulin-suppressed proximal tubule gluconeogenesis is mediated via the IRS1/Akt2/mTORC1/2 pathway. Thus, distinct pathways may play important roles in hypertension and hyperglycemia in metabolic syndrome and diabetes., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

While mitochondrial (mt) genomes are used extensively for comparative and evolutionary genomics, few mt genomes of deep-sea species, including hydrothermal vent species, have been determined. The Genus Lepetodrilus is a major deep-sea gastropod taxon that occurs in various deep-sea ecosystems. Using next-generation sequencing, we determined nearly the complete mitochondrial genome sequence of Lepetodrilus nux, which inhabits hydrothermal vents in the Okinawa Trough. The total length of the mitochondrial genome is 16,353bp, excluding the repeat region. It contains 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region, typical of most metazoan genomes. Compared with other vetigastropod mt genome sequences, L. nux employs a novel mt gene arrangement. Other novel arrangements have been identified in the vetigastropod, Fissurella volcano, and in Chrysomallon squamiferum, a neomphaline gastropod; however, all three gene arrangements are different, and Bayesian inference suggests that each lineage diverged independently. Our findings suggest that vetigastropod mt gene arrangements are more diverse than previously realized., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Despite lifestyle interventions and various types of anti-hypertension agents, hypertension remains difficult to control in some patients with type 2 diabetes. As a noninvasive device-based approach for the treatment of clinic hypertension, we examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on blood pressure (BP) and pulse rate in Japanese subjects with type 2 diabetes and hypertension., Methods: We examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on BP, pulse rate, and pulse pressure in 212 Japanese subjects (82 men and 130 women; mean age±SE, 65±1years) with type 2 diabetes and hypertension (systolic BP>140mmHg). The subjects were treated with anti-hypertension agents., Results: Systolic and diastolic BP, pulse rate, pulse pressure in the 800-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and lower than those of placebo controls. In addition, systolic and diastolic BP, pulse rate, and pulse pressure in the 500-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and systolic BP, pulse rate, and pulse pressure were significantly lower than those of placebo controls., Conclusions: Low-frequency (800kHz or 500kHz) and low-intensity (25mW/cm(2)) ultrasound irradiation to the forearm might have potential usefulness as a therapeutic application for clinic hypertension in subjects with type 2 diabetes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Ascidians are the closest relatives of vertebrates. Their utility in experimental embryology has been well recognized because of their simple mode of embryogenesis to form tadpole larvae with a basal chordate body plan. Approximately two decades of research, including decoding of the Ciona genome, have promoted ascidians as one of the best systems for exploring genome-wide mechanisms of developmental transcriptional control and chordate evolution., (© 2016 Elsevier Inc. All rights reserved.)

Vertebrate mitochondrial (mt) genomes display highly conserved gene order and relatively low evolutionary rates. However, these features are variable in marine invertebrates. Here we present the mt genome of the lingulid brachiopod, Lingula anatina, from Amami Island, Japan, as part of the nuclear genome project. We obtain ~2000-fold coverage of the 17.9-kb mt genome using Illumina sequencing, and we identify hypervariable regions within the same individual. Transcriptome analyses show that mt transcripts are polycistronic and expressed differentially. Unexpectedly, we find that the mt gene order of Amami Lingula is completely shuffled compared to that of a specimen from Yanagawa, suggesting that there may be cryptic species. Using breakpoint distance analyses with 101 metazoan mt genomes, we show that the evolutionary history of mt gene order among lophotrochozoans is unique. Analyses of non-synonymous substitution rates reveal that mt protein-coding genes of Lingula have experienced rapid evolution comparable to that expected for interspecific comparisons. Whole genome phylogenetic analyses suggest that mt genomes have limited value for inferring the phylogenetic positions of lophotrochozoans because of their high evolutionary rates in brachiopods and bivalves., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our previous study indicates that hyperinsulinemia in metabolic syndrome in the absence of nephropathy may promote hypertension by stimulating renal proximal tubule (PT) sodium transport via insulin receptor substrate (IRS) 2/phosphoinositide 3-kinase pathway. In the present study we showed that the stimulatory effect of insulin on the Na(+)-HCO3(-) cotransporter NBCe1 in isolated PTs was completely preserved in type 2 diabetic rats with overt nephropathy. Furthermore, the IRS2 expression and insulin-induced Akt phosphorylation in kidney cortex were preserved in these rats. By contrast, the IRS1 expression in kidney cortex was markedly reduced, which might be relevant to enhanced renal gluconeogenesis consistently reported in diabetes. The stimulatory effect of insulin on NBCe1 was preserved also in a human type 2 diabetic patient with advanced nephropathy. These results revealed that insulin can stimulate PT sodium transport even in type 2 diabetes with overt nephropathy. In addition to hypoglycemia, insulin-induced renal sodium retention might also play a role in increased cardiovascular risk associated with intensive glycemic control in type 2 diabetic patients with nephropathy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hyperinsulinemia can contribute to hypertension through effects on sodium transport. To test whether the stimulatory effect of insulin on renal proximal tubule sodium transport is preserved in insulin resistance, we compared the effects of insulin on abdominal adipocytes and proximal tubules in rats and humans. Insulin markedly stimulated the sodium-bicarbonate cotransporter (NBCe1) activity in isolated proximal tubules through the phosphoinositide 3-kinase (PI3-K) pathway. Gene silencing in rats showed that while insulin receptor substrate (IRS)1 mediates the insulin effect on glucose uptake into adipocytes, IRS2 mediates the insulin effect on proximal tubule transport. The stimulatory effect of insulin on glucose uptake into adipocytes was severely reduced, but its stimulatory effect on NBCe1 activity was completely preserved in insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats and patients with insulin resistance. Despite widespread reduction of IRS1 and IRS2 expression in insulin-sensitive tissues, IRS2 expression in the kidney cortex was exceptionally preserved in both OLETF rats and patients with insulin resistance. Unlike liver, acute insulin injection failed to change the expression levels of IRS2 and sterol regulatory element-binding protein 1 in rat kidney cortex, indicating that regulatory mechanisms of IRS2 expression are distinct in liver and kidney. Thus, preserved stimulation of proximal tubule transport through the insulin/IRS2/PI3-K pathway may play an important role in the pathogenesis of hypertension associated with metabolic syndrome.

The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin·serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D2 and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Postprandial hypertriglyceridemia is a risk factor for atherosclerotic disease. However, the postprandial triglyceride (PTG) concentration fluctuates markedly and is poorly reproducible. The aim of this study was to determine whether the mean PTG (mean-PTG) concentration is a risk factor for carotid atherosclerosis in patients with type 2 diabetes., Methods: We measured the fasting and postprandial lipid concentrations, and the maximum intima-media thickness (max IMT) of carotid arteries by ultrasound in 115 diabetic patients. A carotid plaque was defined as max IMT of >1.0mm. The mean-PTG concentration was calculated from several PTG concentrations measured on different days during a 1-year follow-up period., Results: PTG concentrations showed marked intra-individual variability, and ranged from 0.29 to 6.03 mmol/l. Patients with carotid plaques had higher mean-PTG concentrations than those without carotid plaques (1.51 ± 0.57 vs. 1.29 ± 0.47 mmol/l, p=0.025). Neither fasting triglycerides nor one-point PTG concentrations differed between the two groups. Multivariate stepwise logistic regression analysis revealed that the mean-PTG concentration was significantly associated with carotid plaques [OR 1.20 (95% CI, 1.05-1.37), p=0.009], even after adjusting for traditional risk factors including HDL-cholesterol, LDL-cholesterol, age, hypertension, and duration of diabetes., Conclusions: The mean-PTG concentration is an independent risk factor for carotid atherosclerosis in patients with type 2 diabetes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Several studies have suggested that measurement of high-density lipoprotein (HDL) 2 and HDL3 subfractions might be more useful for evaluating coronary risk than total HDL-cholesterol (C). However, methods of measuring HDL2 and HDL3 are quite laborious for general clinical use. Development of a quick and easy method of measuring HDL subfractions has been long-awaited., Methods: Triglyceride (TG) rich lipoproteins (TRLs), low-density lipoprotein (LDL), HDL2, and HDL3 were used for screening of surfactants and enzymes to react selectively with HDL3-C and to decompose other lipoproteins., Results: In order to develop HDL3-C homogeneous assay, polyoxyethylene styrenated phenyl ether derivative, for which the hydrophilic lipophilic balance (HLB) value is 13.6, was adopted as the most effective and specific surfactant for selection of HDL3 from HDL. Sphingomyelinase (SMase) reacted with TRLs and LDL preferentially, and decomposed them. HDL2-C was estimated by subtracting measured HDL3-C from total HDL-C, directly measured by homogeneous method. The homogeneous assay exhibited excellent correlations with the results of HDL3-C and HDL2-C measured by standard ultracentrifugation (R(2)=0.848 and 0.982, respectively)., Conclusions: We established a rapid and effective, fully-automated assay for the measurement of HDL3-C. Furthermore, the subtraction of HDL3-C from total HDL-C allows concurrent determination of HDL2-C., (© 2013.)

Background: Although the circulating levels of remnant-like particle cholesterol (RLP-C) or serum amyloid A-low-density lipoprotein (SAA-LDL) can individually be increased in subjects with metabolic syndrome (MetS), the correlation between the two markers has not yet been previously studied. In the present study, we aimed to investigate the correlation between RLP-C and SAA-LDL in obese subjects with MetS in comparison to those without MetS., Methods: A total of 436 obese subjects were divided into groups with MetS and without MetS (male/female 75/143, mean age 49 years, current smokers 16% in both groups) by applying the age-, gender-, and smoking habit-matching method based on the database in the multicenter Japan Obesity and Metabolic Syndrome Study (JOMS). The data, including RLP-C and SAA-LDL, were compared in each group., Results: Significantly greater levels of RLP-C or SAA-LDL were observed in subjects with MetS in comparison with those without MetS. There was a significantly positive correlation between RLP-C and SAA-LDL, with a relatively greater correlation in subjects with MetS (coefficient = 0.290, P < .01) in comparison with those without MetS (coefficient = 0.181, P < .01). Multivariate-adjusted correlation analyses showed a greater correlation between RLP-C and SAA-LDL in subjects with MetS, relative to those without MetS, although the significant correlation decreased in both groups when the hypertriglyceridemic states were taken into account., Conclusions: A relatively greater and positive correlation between greater levels of RLP-C and SAA-LDL in obese subjects with MetS, in comparison with those without MetS, may be linked to the development of MetS-related cardiovascular disease., (Copyright © 2011 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-β-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The incidence of neurological deficits is reportedly low after sacrificing the affected nerve root during spinal schwannoma treatment. Although the incidence has been widely reported, the operative method for nerve root resection has been not clarified. To evaluate the safety of pure nerve root resection, we focused on solitary spinal schwannomas below the thoracolumbar level and investigated the effect of affected nerve resection., Methods: Twenty-three spinal schwannoma patients were retrospectively examined. The mean age at surgery was 53 years. We investigated preoperative symptoms, duration of the disorder, postoperative neurological deficits, and clinical outcomes. In addition, we measured tumor size on computed tomography after myelography or on magnetic resonance images using image-analysis software. We retrospectively assessed correlations among duration of symptoms, tumor size, and postoperative neurological deficits., Results: The tumors comprised 19 intradural schwannomas and 4 dumbbell-shaped schwannomas. No postoperative neurological deficits were observed in the intradural schwannoma patients. In contrast, three of the four dumbbell-shaped schwannoma patients experienced postoperative neurological deficits. Among these three patients, two recovered quickly whereas one never recovered. The mean duration of the disorder was 29 months. The postoperative modified JOA score (13.0) was significantly improved compared with the preoperative score (8.9). The mean maximum tumor sizes were 97.2 mm(2) for the intradural schwannomas and 884.0 mm(2) for the dumbbell-shaped schwannomas. There were no correlations among tumor size, duration of the disorder, and postoperative neurological deficits., Conclusions: On the basis of this study, we recommend pure single nerve resection for treatment of intradural spinal schwannomas before such tumors progress and involve other normal roots, because postoperative neurological deficits did not occur in our intradural schwannoma patients, irrespective of tumor size, when this procedure was used. However, dumbbell-shaped schwannoma patients should be carefully treated operatively, because high incidence of postoperative neurological deficits can be expected.

Objective: Metabolic syndrome (MetS) is associated with impaired angiogenesis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptor, VEGF receptor-2 (VEGFR-2), whereas the expression of VEGF and VEGFR-2 in the myocardium of insulin-resistant rats is down-regulated. Soluble VEGF receptor-1 (sVEGFR-1) and -2 (sVEGFR-2) have been reported to inhibit angiogenesis both in vitro and in vivo. However, the balance between circulating levels of VEGF and its soluble receptors, which may reflect and/or affect cardiovascular VEGF signaling, in subjects with MetS is unknown., Methods and Results: We carried out a cross-sectional study including 272 consecutive, apparently healthy subjects who were not receiving any drugs. Plasma levels of VEGF and serum levels of its soluble receptors were determined using enzyme-linked immunosorbent assays. VEGF and sVEGFR-1 levels did not differ between subjects with and those without MetS. However, sVEGFR-2 levels were significantly increased in MetS compared with non-MetS subjects. Stepwise regression analysis revealed that HOMA-IR was the strongest independent determinant of the sVEGFR-2 level. Accordingly, the mean sVEGFR-2 levels increased in proportion to both the accumulation of components of MetS and quartile of HOMA-IR. Interestingly, multiple regression analyses revealed that independent determinants of VEGF were the body mass index and blood pressure, whereas, in contrast, those of sVEGFR-2 were HOMA-IR and high-sensitivity C-reactive protein., Conclusions: The correlation of sVEGFR-2 with insulin resistance supports the need for further investigations to define the clinical utility and predictive value of serum sVEGFR-2 levels in cardiovascular dysfunction in MetS., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3'UTRs. Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3'UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations. We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.

Background: Raloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts' differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation., Methods: Female and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 10-6 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed., Results: On histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects., Conclusions: 10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

Background: The putative association between the novel oxidized low-density lipoprotein markers, serum amyloid A-LDL (SAA-LDL) and alpha1-antitrypsin-LDL (AT-LDL), and obesity and the metabolic syndrome (MetS) has not been previously studied. In the present report, we investigated the levels of SAA-LDL and AT-LDL in relation to the components of the MetS. We also assessed the effect of weight reduction therapy on serum SAA-LDL and AT-LDL levels among obese subjects., Methods: The study population included 421 obese Japanese outpatients (185 men and 236 women, mean age: 51.1 years) enrolled in the multicenter Japan Obesity and Metabolic Syndrome Study (JOMS). The novel oxidized low-density lipoprotein markers, serum SAA-LDL and AT-LDL, were measured in all participants., Results: Circulating SAA-LDL levels were independently associated with the presence and the number of components of the MetS. SAA-LDL levels were also significantly and independently correlated with high-sensitivity C-reactive protein. Notably, successful weight reduction resulted in a significant decrease in circulating SAA-LDL concentrations. Levels of AT-LDL were not associated with the MetS., Conclusions: We documented, for the first time, that serum SAA-LDL levels correlate positively with the number of components of the MetS and weight reduction. Whether SAA-LDL may be involved in the pathophysiology of MetS and atherosclerosis deserves further investigation.

Objective: Dysregulation of tissue-specific intracellular glucocorticoid reactivation is implicated in obesity and related metabolic diseases in humans. The ratio of end products of glucocorticoid metabolism in fresh urine sample, tetrahydrocortisol (THF) + allo-tetrahydrocortisol (allo-THF) vs. tetrahydrocortisone (THE), i.e., the urinary ratio is regarded as an index of the systemic balance underlying intracellular glucocorticoid metabolism, where the enzymes, 11β-hydroxysteroid dehydrogenase type 1 and type 2 as well as 5α- and 5β-reductase are involved in a tissue-specific manner., Methods: To explore the clinical implications of the urinary ratio in obesity and related metabolic diseases, the urinary ratio was determined by gas chromatography and mass spectrometry., Results: The urinary ratio was shown to be constant and reproducible in the same individuals. The ratio was found to inversely correlate with BMI (P < 0.01), waist circumference (P < 0.01), and liver transaminase (P < 0.05) in a large cohort of ∼200 Japanese subjects. This finding suggests that the systemic balance underlying intracellular glucocorticoid reactivation was suppressed in obesity and liver dysfunction. Consistent with this notion, the ratio was decreased in patients with non-alcoholic steatohepatitis (P < 0.01). The urinary ratio was not altered in patients with type 2 diabetes on a 2-month mild calorie restriction. In contrast, the ratio was significantly reduced in patients who responded to the anti-diabetic pioglitazone (P < 0.01)., Conclusion: The present study provides novel evidence that the urinary ratio reflects the facet of adipose tissue and liver function in humans, thereby offering a unique opportunity to evaluate obesity-related diseases., (© 2009 Asian Oceanian Association for the Study of Obesity . Published by Elsevier Ltd. All rights reserved.)

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.

Urinary neutrophil gelatinase-associated lipocalin (Ngal or lipocalin 2) is a very early and sensitive biomarker of kidney injury. Here we determined the origin and time course of Ngal appearance in several experimental and clinically relevant renal diseases. Urinary Ngal levels were found to be markedly increased in lipoatrophic- and streptozotocin-induced mouse models of diabetic nephropathy. In the latter mice, the angiotensin receptor blocker candesartan dramatically decreased urinary Ngal excretion. The reabsorption of Ngal by the proximal tubule was severely reduced in streptozotocin-induced diabetic mice, but upregulation of its mRNA and protein in the kidney was negligible, compared to those of control mice, suggesting that increased urinary Ngal was mainly due to impaired renal reabsorption. In the mouse model of unilateral ureteral obstruction, Ngal protein synthesis was dramatically increased in the dilated thick ascending limb of Henle and N was found in the urine present in the swollen pelvis of the ligated kidney. Five patients with nephrotic syndrome or interstitial nephritis had markedly elevated urinary Ngal levels at presentation, but these decreased in response to treatment. Our study shows that the urinary Ngal level may be useful for monitoring the status and treatment of diverse renal diseases reflecting defects in glomerular filtration barrier, proximal tubule reabsorption, and distal nephrons.

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miR-143 has been proposed to play a role in the differentiation of adipocytes in culture. However, the mechanism regulating the expression of miR-143 in adult adipose tissue during the development of obesity in vivo is unknown. Here in, we showed that the expression of miR-143 in the mesenteric fat was up-regulated in mice fed a high-fat diet. Increased miR-143 expression was associated with an elevated body weight and mesenteric fat weight. Furthermore, miR-143 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma and aP2 as well as plasma levels of leptin, one of the important adipocytokines involved in insulin resistance. These findings provide the first evidence for the up-regulated expression of miR-143 in the mesenteric fat of high-fat diet-induced obese mice, which might contribute to the regulated expression of adipocyte genes involved in the pathophysiology of obesity.

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 microM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-alpha-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 microM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

We have carried out a comprehensive survey of the spatiotemporal expression of cadherin superfamily genes in the basal chordate Ciona intestinalis, as an example of a genome-wide expression study of a gene family directly regulating cellular processes in morphogenesis. We found 15 definitely expressed cadherin superfamily genes in the Ciona intestinalis genome. Up to the late gastrula stage, all identified delta-protocadherins and the type II classical cadherin, but not other subfamily members, were zygotically expressed. At later stages, however, all cadherin superfamily genes were expressed in the nervous system. These data are useful for understanding the role of these genes in Ciona development and the evolution of chordates.

Animal development is controlled by gene regulation networks that are composed of sequence-specific transcription factors (TF) and cell signaling molecules (ST). Although housekeeping genes have been reported to show clustering in the animal genomes, whether the genes comprising a given regulatory network are physically clustered on a chromosome is uncertain. We examined this question in the present study. Ascidians are the closest living relatives of vertebrates, and their tadpole-type larva represents the basic body plan of chordates. The Ciona intestinalis genome contains 390 core TF genes and 119 major ST genes. Previous gene disruption assays led to the formulation of a basic chordate embryonic blueprint, based on over 3000 genetic interactions among 79 zygotic regulatory genes. Here, we mapped the regulatory genes, including all 79 regulatory genes, on the 14 pairs of Ciona chromosomes by fluorescent in situ hybridization (FISH). Chromosomal localization of upstream and downstream regulatory genes demonstrates that the components of coherent developmental gene networks are evenly distributed over the 14 chromosomes. Thus, this study provides the first comprehensive evidence that the physical clustering of regulatory genes, or their target genes, is not relevant for the genome-wide control of gene expression during development.

Tunicates are the only animal group known to synthesize cellulose. The current hypothesis is that a horizontally acquired cellulose synthase gene of bacterial origin might have contributed to the establishment of this unique trait. Cellulose biosynthesis in tunicates thus provides an opportunity to understand how a foreign gene was assimilated into a new genome to establish a new trait. Because little is known of the molecular mechanisms underlying cellulose biosynthesis, we set up a practical assessment of cellulose in the ascidian tunicate Ciona intestinalis. We first demonstrated and characterized cellulose in the tunic of adult specimens by chemical purification and by subsequent scanning electron microscopic observation and X-ray diffractometry. Next, we showed that Fourier transform infrared spectroscopic microscopy (FTIR microscopy) can be used to assess cellulose in the small tunic of individual larval specimens without chemical purification. Using FTIR microscopy together with a blastomere isolation technique, we demonstrated that cellulose biosynthesis occurred cell-autonomously in the animal hemisphere of an embryo where the future epidermis, the known site of cellulose biosynthesis, will arise. We combined FTIR microscopy with morpholino antisense oligonucleotide-mediated gene knockdown technology to generate a reverse genetic system to identify genes involved in cellulose biosynthesis. FTIR microscopy was thus able, in combination with current research resources, to contribute to cellular and molecular investigations of animal cellulose biosynthesis.

Recent whole-genome studies and in-depth expressed sequence tag (EST) analyses have identified most of the developmentally relevant genes in the urochordate, Ciona intestinalis. In this study, we made use of a large-scale oligo-DNA microarray to further investigate and identify genes with specific or correlated expression profiles, and we report global gene expression profiles for about 66% of all the C. intestinalis genes that are expressed during its life cycle. We succeeded in categorizing the data set into 5 large clusters and 49 sub-clusters based on the expression profile of each gene. This revealed the higher order of gene expression profiles during the developmental and aging stages. Furthermore, a combined analysis of microarray data with the EST database revealed the gene groups that were expressed at a specific stage or in a specific organ of the adult. This study provides insights into the complex structure of ascidian gene expression, identifies co-expressed gene groups and marker genes and makes predictions for the biological roles of many uncharacterized genes. This large-scale oligo-DNA microarray for C. intestinalis should facilitate the understanding of global gene expression and gene networks during the development and aging of a basal chordate.

Anural ascidians show embryogenesis during which tail formation does not take place. This mode of development is a derived character acquired several times independently in ascidian evolution. We identified approximately 20,000 each ESTs (i. e. 10,000 clones each were sequenced from both 5' and 3' ends) of adult gonads, cleaving-embryos, gastrulae/neurulae, embryos before hatching, and hatched larvae of the anural ascidian Molgula tectiformis, in order to comprehensively investigate the molecular mechanism of tailless evolution. Analyses of these ESTs showed that in this species, (1) the expression of embryonic/larval muscle structural genes which are expressed abundantly during embryogenesis of the urodele ascidian Ciona intestinalis, is suppressed; (2) genes that encode proteins with no similarity to known proteins of other organisms are abundantly expressed; (3) genes that show similarity with those up-regulated at metamorphosis in urodele ascidians are up-regulated within several hours after hatching; and (4) 15 of 35 putative orthologues of the downstream components of Brachyury, a key transcription factor for ascidian notochord formation, were found in the ESTs, even though differentiation of notochord is suppressed in this species. We discuss these remarkable results that allow insight into the molecular mechanism(s) responsible for the anural mode of ascidian development.

Umbilical cord blood (CB) is a promising source for regeneration therapy in humans. Recently, it was shown that CB was a source of mesenchymal stem cells as well as hematopoietic stem cells, and further that the mesenchymal stem cells could differentiate into a number of cells types of mesenchymal lineage, such as cardiomyocytes (CMs), osteocytes, chondrocytes, and fat cells. Previously, we reported that brown adipose tissue derived cells (BATDCs) differentiated into CMs and these CMs could adapt functionally to repair regions of myocardial infarction. In this study, we examined whether CB mononuclear cells (CBMNCs) could effectively differentiate into CMs by coculturing them with BATDCs and determined which population among CBMNCs differentiated into CMs. The results show that BATDCs effectively induced CBMNCs that were non-hematopoietic stem cells (HSCs) (educated CB cells: e-CBCs) into CMs in vitro. E-CBCs reconstituted infarcted myocardium more effectively than non-educated CBMNCs or CD34-positive HSCs. Moreover, we found that e-CBCs after 3 days coculturing with BATDCs induced the most effective regeneration for impaired CMs. This suggests that e-CBCs have a high potential to differentiate into CMs and that adequate timing of transplantation supports a high efficiency for CM regeneration. This strategy might be a promising therapy for human cardiac disease.

The recent decoding of a number of animal genomes has provided unprecedented information regarding evolution and gene structures, but this information must be supplemented with precise gene annotations and the temporal and spatial expression patterns of individual genes. In the present study, we systematically identified and characterized 566 zinc finger genes in the genome of Ciona intestinalis, an emerging model system for genome-wide studies of development and evolution. Of these genes, 356 genes encoded a potential transcription factor based on putative nucleic acid binding activity or domains of unknown function. We further examined the expression patterns of 225 genes during embryogenesis, and, when considered with a previous study [Imai, K.S., Hino, K., Yagi, K., Satoh, N., Satou, Y., 2004. Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, 4047-4058], we have characterized the developmental expression patterns of nearly 85% of the potential zinc finger-containing transcription factors. Overall, zinc finger genes are preferentially maternally expressed with little larval expression during development. The present study provides a valuable reference for genome-wide studies in this species and for future studies wishing to examine zinc finger gene expression patterns in other animals.

Ascidian larval mesenchyme cells, comprising about 900 cells, are derived from the A7.6, B8.5 and B7.7 blastomere pairs in the 110-cell embryo. Previous studies showed that the properties of mesenchyme cells are not uniform among the three lines in embryos of Ciona savignyi and Ciona intestinalis. After metamorphosis, the larval mesenchyme cells form the mesodermal tissues or organs of the adult body. In the present study, the developmental fates of A7.6-, B8.5- and B7.7-line mesenchyme cells were traced using DiI to determine the origins of juvenile mesodermal tissues of C. savignyi. It was demonstrated that each of the A7.6-, B8.5- and B7.7-line mesenchyme cells is distributed in different positions of the larval trunk, and then give rise to the different mesodermal tissues of juveniles. Twist-like 1 is a transcription factor gene essential for the specification of larval mesenchyme cells. Knockdown of this gene with specific morpholino antisense oligonucleotides affected not only the specification of larval mesenchyme cells, but also the formation of most of the mesodermal tissues of juveniles. The juvenile mesodermal tissues in the Twist-like 1-knockdown specimen were never compensated by the surrounding tissues. The present results therefore indicate that Twist-like 1 is required for the differentiation of most mesodermal precursors of adults.

A novel gene (Ci-Rga) essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis is reported here. This gene was identified through functional screening of Ciona genes required for development by translational inhibition experiments with morpholino antisense oligonucleotides. The deduced protein of Ci-Rga contains two copies of a domain with unknown function called the MtN3/saliva domain. Phylogenetic analysis showed that Ci-Rga belongs to the MtN3/saliva family of genes conserved among metazoans and plants, and is an ortholog of mouse Rga (Recombination-activating gene 1 gene activation). During Ciona embryogenesis, both maternal and zygotic transcripts of Ci-Rga were expressed. Translational inhibition of Ci-Rga with specific morpholino resulted in abnormal embryos in which the cleavage pattern became atypical and expression of marker genes for each of the six major tissues, namely the endoderm, muscle, mesenchyme, notochord, neural tissue, and epidermis, was lost or suppressed at the tailbud stage. Although differentiation of all the six major tissues was affected by Ci-Rga knock-down, the degree of abnormalities and the timing of appearance of abnormalities were different among tissues. Expression analysis of developmentally important genes involved in the fate specification, such as Ci-Bra, Ci-Twist-like1a, Ci-Otx, Ci-Fgf9/16/20, Ci-Lhx3, Ci-FoxD, and Ci-Tbx6b, showed that an initial step of the fate specification of notochord, mesenchyme, and neural tissue, but not of endoderm or muscle, is impaired in the knock-down embryo. These results showed that Ci-Rga is a multifunctional gene essential for tissue differentiation during embryogenesis, and is primarily required for the fate specification of notochord, mesenchyme, and neural tissue, and provide some insights into the function of this little-known group of genes.

The establishment of body axes and specification of early embryonic cells depend on maternally supplied transcripts and/or proteins, several of which are localized at specific regions of fertilized eggs and early embryos. The ascidian is known to exhibit a mosaic mode of development, and this mode is largely dependent on localized maternal factors. Using blastomere isolation, microarray and whole-mount in situ hybridization, the present study of Ciona intestinalis demonstrates that maternal transcripts of a total of 17 genes are localized at the posterior-most region of fertilized eggs and early embryos. Ten of them are newly identified in the present study, while the remaining seven genes have already been characterized in previous studies. In addition, maternal transcripts of two genes, in addition to 14 genes encoded by the mitochondrial genome, showed a mitochondria-like distribution. Despite the present comprehensive approach, we could not identify maternal transcripts that are clearly localized to the animal-pole side, the vegetal-pole side, the anterior-side or other specific regions of the early embryo. Therefore, we concluded that the posterior-most localization and mitochondria-like distribution appear to be major specialized patterns of maternal transcripts in early Ciona embryos.

Nitric oxide (NO) is a potent regulator in the cardiovascular system; it is generated by the nitric oxide synthase (NOS) family of proteins. NO produced in endothelial cells plays a crucial role in vascular functions. The aim of this study was to clarify the effect of diabetes on aortic NO synthesis in a model of genetic hypertension and determine whether captopril modulates this effect. Diabetes was induced in ten weeks old spontaneously hypertensive rats (SHR) by streptozotocin injection. The rats were allocated into 3 groups: control group 1, non-diabetic SHR; group 2, diabetic SHR; group 3, diabetic SHR group receiving captopril at 80 mg/kg in drinking water for 4 weeks. Mean blood pressure (MBP) was measured once a week by tail-cuff method. Aortic NO metabolities (nitrite/nitrate) and endothelial NOS (NOS-3) were assayed by Griess reaction and by immunoblotting and immunohistochemistry, respectively. There was a significant decrease in nitrite/nitrate (NOx) in aortas of diabetic SHR compared with controls. The decrease of aortic NOx in diabetic SHR was accompanied by a decrease in NOS-3 expression. Captopril treatment reduced MBP without affecting either NOx level or NOS-3 expression in aortas of diabetic SHR. We conclude that STZ-induced diabetes decreased NO in aortas of SHR that may reflect endothelial cell dysfunction; captopril administration decreased MBP without affecting NO level in aortas of diabetic SHR which suggest that the blood pressure-lowering effects of captopril were independent of NO.

Maternally deposited mRNA encoding the Zic family zinc-finger protein Ci-macho1 is a determinant responsible for muscle cell differentiation in Ciona intestinalis embryos. In a previous study, we identified possible Ci-macho1 downstream genes, which include seven transcription factor genes and seven signaling molecule genes (Yagi, K., Satoh, N., Satou, Y., 2004. Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1. Dev. Biol. 274, 478-489), suggesting complex Ci-macho1 downstream cascades. Here, we show that of the Ci-macho1 downstream genes, only Ci-Tbx6b and Ci-Tbx6c promote ectopic differentiation of muscle cells when misexpressed in non-muscle blastomeres. Overexpression of Ci-Tbx6b or Ci-Tbx6c in Ci-macho1 knockdown embryos is able to compensate for the functional loss of Ci-macho1 and promote differentiation of muscle cells. In addition, we show that knockdown of each of Ci-Tbx6b or Ci-Tbx6c suppresses the initiation of muscle protein gene expression, and both gene products appear to recognize a similar binding sequence. However, later expression of muscle protein genes at the tailbud stage is only reduced in Ci-Tbx6b knockdown embryos and undisturbed in Ci-Tbx6c knockdown embryos. Although ectopic expression or knockdown of Ci-ZicL alone does not affect muscle cell differentiation, simultaneous knockdown of Ci-Tbx6b, Ci-Tbx6c, and Ci-ZicL completely abolishes muscle cell differentiation, as in the case of knockdown of Ci-macho1 and Ci-ZicL. These results strongly suggest that muscle cell differentiation in Ciona embryos is controlled by four key factors: maternal macho1 and zygotic Tbx6b, Tbx6c, and ZicL. The two T-box genes are primary mediators of macho1 function, and cooperation between the zygotically expressed transcription factors is indispensable for muscle cell differentiation in Ciona embryos.

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.

Autonomous differentiation of primary muscle cells in ascidian embryos is triggered by a maternal determinant recently identified as the macho-1 gene. macho-1 encodes a transcription factor of the Zic family with five C2H2 zinc-finger motifs. In the present study, we firstly performed a screen, using a quantitative PCR method, of genes encoding transcription factors and components in major signaling pathways to identify those regulated downstream of Ci-macho1 in early embryos of Ciona intestinalis. The amount of transcripts for a total of 64 genes was altered at the 32-cell stage depending on the Ci-macho1 activity level. Whole-mount in situ hybridization assays revealed that the alteration of expression for at least 13 of them was adequately visualized to confirm the results of quantitative PCR. Second, we determined a possible binding sequence of Ciona macho1. macho1 recombinant proteins of both C. intestinalis and Ciona savignyi recognized a sequence, 5'-GCCCCCCGCTG-3', that resembles the mammalian Zic binding site. In addition, most of the genes identified as potential Ci-macho1 downstream genes, in particular Ci-Tbx6b and Ci-snail, possessed plausible Ci-macho1-binding sequences in their 5' upstream region, suggesting their direct activation by Ci-macho1. Furthermore, some of the genes including three Wnt genes noted in the quantitative analyses implied that Ci-macho1 is involved in the differentiation of endoderm and mesenchyme via intracellular communications.

The ascidian embryonic mesenchyme, comprising about 900 cells, forms mesodermal tissues or organs of the adult body after metamorphosis. The mesenchyme originates from the A7.6 [trunk lateral cells (TLCs)], B7.7, and B8.5 blastomeres of the 110-cell stage embryo. Previous studies showed that FGF9/16/20 is required for specification of the mesenchyme in Ciona embryos and that two different (A7.6 and B8.5/B7.7) but partially overlapping molecular mechanisms are associated with the expression of a basic helix-loop-helix (bHLH) transcription factor gene, Twist-like1, in the mesenchymal precursors, which triggers the differentiation process of mesenchyme cells. In the present study, we examined whether the three embryonic lineages express the same mesenchyme-specific structural genes under the control of a common mechanism or whether the three lineages are characterized by the expression of genes specific to each of the lineages. We characterized nine mesenchyme-specific genes in Ciona embryos and found that five were expressed in A7.6/B8.5/B7.7, two in B8.5/B7.7, and two in B7.7 only. FGF9/16/20 and Twist-like1 were required for the expression of all the mesenchyme-specific genes, except for three A7.6/B8.5/B7.7-specific genes in A7.6 progenitors. Overexpression of FGF9/16/20 or Twist-like1 upregulated the expression of A7.6/B8.5/B7.7- and B8.5/B7.7-specific genes, while it downregulated the expression of B7.7-specific genes. These results provide evidence that the differentiation of each of the three mesenchyme lineages of Ciona embryos is characterized by the expression of a specific set of genes, whose expression is controlled differentially.

To analyze global gene expressions, we constructed a cDNA microarray from a basal chordate, the ascidian Ciona intestinalis. Ciona is a cosmopolitan species and a genomic analysis of Ciona revealed that ascidians had approximately 15,500 protein-coding genes. Our "Ciona intestinalis cDNA chip version 1 (Ci cDNA chip ver. 1)" has arrayed 13,400 unique Ciona cDNAs. To establish a detection system for gene expression profiles in wild ascidians using a cDNA microarray, we analyzed gene expressions in the whole body of Ciona adults after exposure to 100 nM tributyltin (TBT) for 24 h. In our preliminary array data using Ci cDNA chip ver. 1, we found more than 200 genes that showed strong differential expressions. These genes encoded proteins that were concerned with stress response, detoxification, oxidoreduction reaction, biosynthesis, and catabolism. This, the first large cDNA microarray of this animal, should facilitate analyses of global gene expressions following exposure to TBT.

Approximately 4% of the Japanese population genetically lack plasma platelet activating factor acetylhydrolase (PAF-AH) and show a higher prevalence of thromboembolic disease, but whether they are susceptible to another PAF-related disease, asthma, remains controversial. To determine the role of plasma PAF-AH in airway physiology, we performed PAF bronchoprovocation tests in 8 plasma PAF-AH-deficient subjects and 16 control subjects. Serial inhalation of PAF (1-1000 microg/ml) concentration-dependently induced acute bronchoconstriction, but there was no significant difference between PAF-AH-deficient and control subjects (11.7 +/- 4.6% vs. 9.6 +/- 2.8% decrease in forced expiratory volume in 1 s). Transient neutropenia after single inhalation of PAF (1000 microg/ml) showed no significant difference between the groups either in its magnitude (72 +/- 11% vs. 65 +/- 9% decrease) or duration (4.1 +/- 1.0 vs. 3.3 +/- 0.8 min). In conclusion, a lack of plasma PAF-AH activity alone does not augment physiological responses to PAF in the airway.

Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms.