Tohru Suzuki

Publications (41)79.93 Total impact

• Article: Molecular characterization and expression analysis of chitinase from the Pacific oyster Crassostrea gigas.

  Yuki Okada, Kunihiro Yamaura, Tohru Suzuki, Naoki Itoh, Makoto Osada, Keisuke G Takahashi
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  ABSTRACT: Chitinases are necessary enzymes supporting functions as a host defense factor against chitin-coated pathogens. They also function as a digestive enzyme for the hydrolysis of dietary chitin. We conducted characterization and assessed the tissue expression of the encoding gene of a chitinase (EC 3.2.1.14), Cg-Chit1, and the production of recombinant protein of Cg-Chit1, from the Pacific oyster, Crassostrea gigas. Chitinase activity in mantle extracts was detected to a marked degree in samples collected in July and August. Mantle chitinase worked well at pH5.5, 7.0, and 8.5 tested in this study. RT-PCR showed that Cg-Chit1expression is highly tissue-specific in the hemocytes and mantle. We then determined the distribution of Cg-Chit1 mRNA in C. gigas hemocytes and mantle histologically using in situ hybridization. Of the two subgroups of oyster hemocytes, granulocytes (main phagocytes) and hyalinocytes, only the former were found to express Cg-Chit1. In the mantle, chitinase-2 was expressed at the inner lobe of the mantle edge. Recombinant Cg-Chit1 clearly showed chitinase activity in a wide range of neutral/basic pH. These findings suggest that Cg-chit1 functions as a host defense factor to hydrolyze chitin-coated organisms after phagocytosis by granulocytes and to exclude foreign substances from the mantle cavity.
  Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 03/2013; · 1.61 Impact Factor
• Article: Ocular-Side Lateralization of Adult-Type Chromatophore Precursors: Development of Pigment Asymmetry in Metamorphosing Flounder Larvae.

  Youhei Washio, Masato Aritaki, Yuichiro Fujinami, Daisuke Shimizu, Hayato Yokoi, Tohru Suzuki
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  ABSTRACT: The adult-type chromatophores of flounder differentiate at metamorphosis in the skin of ocular side to establish asymmetric pigmentation. In young larva and before metamorphosis, adult-type melanophores that migrate to the ocular side during metamorphosis reside at the base of the dorsal fin as latent precursors. However, the migration route taken by these precursor cells and the mechanisms by which lateralization and asymmetric pigmentation develop on the ocular side are unknown. To further investigate this migration and lateralization, we used in situ hybridization with gch2 probe, a marker for melanoblasts and xanthoblasts (precursors of adult type chromatophores), to examine the distribution of chromatophore precursors in metamorphosing larvae. The gch2-positive precursors were present in the myoseptum as well as in the skin. This finding indicated that these precursors migrated from the dorsal part of the fin to the skin via the myoseptum. Additionally, there were much fewer gch2-positive cells in the myoseptum of the blind side than in the skin and myoseptum of the ocular side, and this finding indicated either that migration of the precursor cells into the myoseptum of blind side was inhibited or that the precursors were eliminated from the myoseptum of the blind side. Therefore, we propose that the signals responsible for development of asymmetric pigmentation in flounder reside not only in the skin but on a larger scale and in multiple tissues throughout the lateral half of the trunk. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:1-15, 2013. © 2013 Wiley Periodicals, Inc.
  Journal of Experimental Zoology Part B Molecular and Developmental Evolution 02/2013; · 2.42 Impact Factor
• Article: Circadian pacemaker in the suprachiasmatic nuclei of teleost fish revealed by rhythmic period2 expression.

  Nanako Watanabe, Kae Itoh, Makoto Mogi, Yuichiro Fujinami, Daisuke Shimizu, Hiroshi Hashimoto, Susumu Uji, Hayato Yokoi, Tohru Suzuki
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  ABSTRACT: In mammals, the role of the suprachiasmatic nucleus (SCN) as the primary circadian clock that coordinates the biological rhythms of peripheral oscillators is well known. However, in teleosts, it remains unclear whether the SCN also functions as a circadian pacemaker. We used in situ hybridization (ISH) techniques to demonstrate that the molecular clock gene, per2, is expressed in the SCN of flounder (Paralichthys olivaceus) larvae during the day and down-regulated at night, demonstrating that a circadian pacemaker exists in the SCN of this teleost. The finding that per2 expression in the SCN was also observed in the amberjack (Seriola dumerili), but not in medaka (Oryzias latipes), implies that interspecific variation exists in the extent to which the SCN controls the circadian rhythms of fish species, presumably reflecting their lifestyle. Rhythmic per2 expression was also detected in the pineal gland and pituitary, and aperiodic per2 expression was observed in the habenula, which is known to exhibit circadian rhythms in rodents. Since the ontogeny of per2 expression in the brain of early flounder larvae can be monitored by whole mount ISH, it is possible to investigate the effects of drugs and environmental conditions on the functional development of circadian clocks in the brain of fish larvae. In addition, flounder would be a good model for understanding the rhythmicity of marine fish. Our findings open a new frontier for investigating the role of the SCN in teleost circadian rhythms.
  General and Comparative Endocrinology 06/2012; 178(2):400-7. · 3.27 Impact Factor
• Article: Continuous illumination through larval development suppresses dopamine synthesis in the suprachiasmatic nucleus, causing activation of α-MSH synthesis in the pituitary and abnormal metamorphic skin pigmentation in flounder.

  Kae Itoh, Youhei Washio, Yuichiro Fujinami, Daisuke Shimizu, Susumu Uji, Hayato Yokoi, Tohru Suzuki
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  ABSTRACT: In order to better understand the endocrine aberrations related to abnormal metamorphic pigmentation that appear in flounder larvae reared in tanks, this study examined the effects of continuous 24-h illumination (LL) through larval development on the expression of tyrosine hydroxylase-1 (th1), proopiomelanocortin (pomc), α-melanophore-stimulating hormone (α-MSH) and melanin concentrating hormone (MCH), which are known to participate in the control of background adaptation of body color. We observed two conspicuous deviations in the endocrine system under LL when compared with natural light conditions (LD). First, LL severely suppressed th1 expression in the dopaminergic neurons in the anterior diencephalon, including the suprachiasmatic nucleus (SCN). Second, pomc and α-MSH expression in the pars intermedia melanotrophs was enhanced by LL. Skin color was paler under LL than LD before metamorphic pigmentation, and abnormal metamorphic pigmentation occurred at a higher ratio in LL. We therefore hypothesize that continuous LL inhibited dopamine synthesis in the SCN, which resulted in up-regulation of pomc mRNA expression in the melanotrophs. In spite of the up-regulation of pomc in the melanotrophs, larval skin was adjusted to be pale by MCH which was not affected by LL. Accumulation of α-MSH in the melanotrophs is caused by uncoupling of α-MSH synthesis and secretion due to inhibitory role of MCH on α-MSH secretion, which results in abnormal metamorphic pigmentation by affecting differentiation of adult-type melanophores. Our data demonstrate that continuous illumination at the post-embryonic stage has negative effects on the neuroendocrine system and pituitary in flounder.
  General and Comparative Endocrinology 02/2012; 176(2):215-21. · 3.27 Impact Factor
• Article: Embryogenic staging of fugu, Takifugu rubripes, and expression profiles of aldh1a2, aldh1a3 and cyp26a1.

  Susumu Uji, Tadahide Kurokawa, Hisashi Hashimoto, Tohru Kasuya, Tohru Suzuki
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  ABSTRACT: Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis - the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.
  Embryologia 06/2011; 53(5):715-25. · 2.21 Impact Factor
• Source

  Available from: PubMed Central

  Article: Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence.

  Yohei Matsuda, Yoshie Ito, Hisashi Hashimoto, Hayato Yokoi, Tohru Suzuki
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  ABSTRACT: Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg) in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.
  Reproductive Biology and Endocrinology 01/2011; 9:45. · 2.05 Impact Factor
• Article: Effects of 3,4-dichloroaniline on expression of ahr2 and cyp1a1 in zebrafish adults and embryos.

  Yoshie Ito, Youhei Matsuda, Tohru Suzuki
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  ABSTRACT: Arylhydrocarbon receptor (Ahr) and cytochrome P4501a1 (Cyp1a1) are members of the Ahr/Cyp1a1 pathway that oxygenates various toxic chemicals including aryl hydrocarbons. To elucidate Ahr/Cyp1a1 pathway responses in teleost fish tissues, we examined the effects of 3,4-dichloroaniline (3,4-DCA), a reference toxic compound known to activate the Ahr/Cyp1a1 pathway, on the expression of arh and cyp1a1 in zebrafish tissues and embryos by means of in situ hybridization (ISH). Our ISH analysis showed that cyp1a1 expression was markedly activated by 3,4-DCA in the gill and intestinal epithelia, skin epidermis, and liver parenchymal cells of adult zebrafish. Before differentiation of the gill, intestine, and liver, skin was the site of cyp1a1 activation in embryos. Unlike the cyp1a1 response, 3,4-DCA-mediated ahr activation was not marked in either adults or embryos, indicating a possibility that stable ahr transcripts persist in the cytoplasm of these cells to induce cyp1a1. Young oocytes (previtellogenic to early vitellogic stage) express ahr; however activation of cyp1a1 by 3,4-DCA was negligible in these oocytes, suggesting that ahr expression in oocytes is not directly linked to cyp1a1 activation. Based on our finding that skin epidermis up-regulates cyp1a1 in response to 3,4-DCA, we demonstrated that fin explants, which can be harvested without sacrificing fish, can be used as a standard for assaying cyp1a1 activation in addition to embryos that are now used.
  Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2010; 152(2):189-94. · 2.62 Impact Factor
• Article: Three members of the iodothyronine deiodinase family, dio1, dio2 and dio3, are expressed in spatially and temporally specific patterns during metamorphosis of the flounder, Paralichthys olivaceus.

  Kae Itoh, Kohei Watanabe, Xiaoming Wu, Tohru Suzuki
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  ABSTRACT: Flounder metamorphosis, marked by eye migration, lateralized pigmentation, and tissue differentiation in the stomach and skeletal muscle, is stimulated by thyroid hormone (TH). It is known that tri-iodothyronine (T3) produced by iodothyronine deiodinase type-1 (Dio1) from thyroxine (T4) enters the blood, whereas T3 produced by Dio2 penetrates into the nucleus of the Dio2-expressing cells, and then Dio3 inactivates both T4 and T3. To better understand the distinct functions of these three deiodinases in T3 regulation during flounder metamorphosis, we examined the tissue expression patterns of dio1, dio2, and dio3 in larvae of the Japanese flounder, Paralichthys olivaceus, by section in situ hybridization (SISH). We found that each deiodinase is expressed in a spatially and temporally specific pattern. dio1 is expressed in liver parenchymal cells from pro-metamorphosis to early climax, while dio2 is expressed in limited regions of the eyes, tectum, and skeletal muscles from pro-metamorphosis to post-climax. Considering these findings together with reports on other vertebrates, we predict that the liver cells expressing dio1 supply T3 to the blood, and that this systemic T3 synchronizes metamorphosis of differentiating tissues throughout the larval body, whereas the eyes, tectum, and skeletal muscles autonomously produce additional T3 for local tissue differentiation. Finally, dio3 expression is detected in skeletal muscle and gastric gland blastemas, which both undergo marked tissue differentiation at metamorphic climax. We hypothesize that dio3 expression protects these tissues from basal T3 levels early in metamorphosis, ensuring, together with the T3 surge from the liver, the synchronization of tissue differentiation at metamorphic climax.
  ZOOLOGICAL SCIENCE 07/2010; 27(7):574-80. · 0.95 Impact Factor
• Article: Muscle development in the Japanese flounder, Paralichthys olivaceus, with special reference to some of the larval-specific muscles.

  Susumu Uji, Tadahide Kurokawa, Tohru Suzuki
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  ABSTRACT: We investigated muscle development in the Japanese flounder Paralichthys olivaceus, focusing primarily on the cranial muscles, using a whole mount immunohistochemical staining method. It is well established that during the very early stages of morphogenesis, until 4 days post hatching (dph), muscles required for feeding develop. Later, between 8 and 16 dph, the muscle composition in the dorsal branchial arches changes to the adult form. We discovered the presence of larval-specific muscles in this ontogenetic period, termed the larval branchial levators 2 and 3, located in the dorsal branchial arches. The larval branchial levators 2 and 3 disappear during the course of development, whereas the others remain as levator internus 1 and levator posterior, which have also been described in adult fish. In place of these regressed muscles, the levatores externi and levator internus 2 develop and regulate the branchial arches. In addition, we found that the levator posterior, which is thought to represent the fifth levator externus, and the levatores externi exhibit different origins. We also found that at least a part of the caudal fin musculature develops from the trunk myotome.
  Journal of Morphology 02/2010; 271(7):777-92. · 1.54 Impact Factor
• Article: Metamorphic pitx2 expression in the left habenula correlated with lateralization of eye-sidedness in flounder.

  Tohru Suzuki, Youhei Washio, Masato Aritaki, Yuichiro Fujinami, Daisuke Shimizu, Susumu Uji, Hisashi Hashimoto
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  ABSTRACT: The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.
  Embryologia 10/2009; 51(9):797-808. · 2.21 Impact Factor
• Article: Induction of Bent Cartilaginous Skeletons and Undulating Notochord in Flounder Embryos by Disulfiram and α, α'-Dipyridyl

  Tohru Suzuki, Tadahide Kurokawa, AnS. Srivastava
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  ABSTRACT: Disulfiram causes undulation of the notochord and bending of pharyngeal cartilages in fish embryos. Using flounder embryos, this study aimed to elucidate which process of pharyngeal arch development was affected by the drug. Since disulfiram is known to block the synthesis of retinoic acid (RA) in vivo, we first examined whether the drug suppresses the expression of Hoxd-4 and shh, RA responsive genes related with the pharyngeal arch development. Disulfiram at a concentration which induces undulation of the notochord and bending of cartilage elements did not affect the expression of these genes. On the other hand, similar phenotypes of anomalies were found to be reproduced both in the notochord and pharyngeal cartilages by α, α′-dipyridyl which reduces the mechanical stability of collagen. Thus, we suppose that disulfiram causes anomalies by decreasing the mechanical stability of collagen and not by suppressing the expression of RA-responsive genes. Since disulfiram blocks ascorbate dehydrogenase, it is our hypothesis that this drug inhibits the maturation of collagen by affecting ascorbic acid metabolism.
  ZOOLOGICAL SCIENCE 08/2009; · 0.95 Impact Factor
• Article: Adult-type pigment cells, which color the ocular sides of flounders at metamorphosis, localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae.

  Kohei Watanabe, Youhei Washio, Yuichiro Fujinami, Masato Aritaki, Susumu Uji, Tohru Suzuki
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  ABSTRACT: Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.
  Embryologia 01/2009; 50(9):731-41. · 2.21 Impact Factor
• Article: Genomic characterization and tissue distribution of leptin receptor and leptin receptor overlapping transcript genes in the pufferfish, Takifugu rubripes.

  Tadahide Kurokawa, Koji Murashita, Tohru Suzuki, Susumu Uji
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  ABSTRACT: Full-length cDNAs encoding the leptin receptor (tfLEPR), leptin receptor overlapping transcript (tfLEPROT) and leptin receptor overlapping transcript-like 1 (tfLEPROTL1) were cloned and sequenced from the pufferfish, Takifugurubripes. The tfLEPR gene encoded an 1116-amino acid protein that includes almost all functionally important domains conserved among vertebrate LEPR such as three fibronectin type III domains, the immunoglobulin (Ig) C2-like domain and a pair of repeated tryptophan/serine motifs. The tfLEPR mRNA was abundantly expressed in the pituitary and ovary and moderately expressed in brain, eye, heart, kidney, liver and testis. Both tfLEPROT and tfLEPROTL1 genes encoded a 130-amino acid protein. Human LEPR gene shares the first and second exons with the LEPROT gene, and they are continuously located on chromosome 1p31. In contrast, TakifuguLEPR and LEPROT were located at different regions of the chromosome. However, both Takifugu regions showed genomic synteny with the human genome around LEPR gene on chromosome 1p31. This result could mean that the Takifugu chromosomes around LEPR and LEPROT genes are paralogous genomic regions derived from genome duplication early in the teleost lineage and the overlapping LEPR and LEPROT genes were subsequently lost.
  General and Comparative Endocrinology 07/2008; 158(1):108-14. · 3.27 Impact Factor
• Article: Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas.

  Kunihiro Yamaura, Keisuke G Takahashi, Tohru Suzuki
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  ABSTRACT: As an initial step in the functional analysis of lectins in the Pacific oyster, Crassostrea gigas, we attempted to obtain the full coding sequences of C. gigas lectins and conduct tissue expression analyses. To obtain lectin genes quickly, we identified C. gigas expressed sequence tags that coded for lectins in GenBank, and selected three encoding partial sequences of C-type lectin 1 (CgCLec-1), galectin (CgGal) and fucolectin. We obtained full open reading frames of CgCLec-1 and CgGal cDNAs by RACE-PCR. CgCLec-1 is a typical C-type lectin with a signal peptide and C-type lectin domain. CgCLec-1 mRNA was expressed only in specialized basophilic cells involved with digestive enzyme secretion in the digestive gland, suggesting that CgCLec-1 is secreted into the lumen of the digestive diverticula. CgGal is a prototype galectin with a single galactose-binding domain that was expressed in all of the tissues examined. As suggested for vertebrate galectin-1 (prototype galectin), CgGal may function in general cell activities such as cell adhesion. Fucolectin in C. gigas was expressed specifically in the gonads, indicating a possible function in gonadal development. CgCLec-1 and CgGal expression in hemocytes was not upregulated after injecting Vibrio tubiashii into adductor muscle, suggesting that bacterial infection does not induce synthesis of these lectins. Of the three lectins examined, CgCLec-1 is an interesting target for future investigations of innate immunity in the digestive system of C. gigas.
  Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 02/2008; 149(1):168-75. · 1.92 Impact Factor
• Article: Identification, cDNA cloning, and mRNA localization of a zebrafish ortholog of leukemia inhibitory factor.

  Takashi Abe, Tsuyoshi Mikekado, Satoru Haga, Yuya Kisara, Kohei Watanabe, Tadahide Kurokawa, Tohru Suzuki
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  ABSTRACT: Leukemia inhibitory factor (LIF) maintains embryonic stem cells in an undifferentiated state. To establish stable cultures of zebrafish embryonic stem cells, recombinant zebrafish LIF (zLIF) is needed because the LIF sequence varies greatly between species. In the current study, we identified the zebrafish (Danio rerio) and pufferfish (Tetraodon nigroviridis) orthologs of lif from genomic databases, and we isolated a cDNA encoding zLIF. Synteny analysis and comparison of sequences identified zebrafish and Tetraodon orthologs of human LIF. The cDNA for zLIF encoded a predicted 215-amino acid protein with a putative 32-amino acid signal peptide, two disulfide bonds, and two N-linked glycosylation sites. We found that transcription of zlif starts at the hatching period during embryogenesis and is present in the brain, visceral organs, bone, and skin.
  Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 06/2007; 147(1):38-44. · 1.92 Impact Factor
• Article: Embryogenesis and expression profiles of charon and nodal-pathway genes in sinistral (Paralichthys olivaceus) and dextral (Verasper variegatus) flounders.

  Hisashi Hashimoto, Masato Aritaki, Kaori Uozumi, Susumu Uji, Tadahide Kurokawa, Tohru Suzuki
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  ABSTRACT: Although it is well known that flounder form external asymmetry by migration of one eye at metamorphosis, the control system that forms this asymmetry is unknown. To help elucidate this mechanism, we here describe the embryogenesis and expression profiles of the Nodal-pathway genes in the Japanese flounder, Paralichthys olivaceus. We also perform a comparative study of the laterality of the expression of these genes in sinistral (P. olivaceus) and dextral (Verasper variegatus) flounders. In P. olivaceus, Kupffer's vesicle forms at the 2-somite stage, after which left-sided expression of spaw starts at the 8-somite stage. Left-sided expression of pitx2 occurs in the gut field at the 15-somite to high-pec stages, in the heart field at the 21-somite stage, and in the dorsal diencephalon at the 27-somite to high-pec stages. In response to left-sided pitx2 expression, the heart, gut, and diencephalon begin asymmetric organogenesis at the pharyngula (heart) and the long-pec (gut and diencephalon) stages, whereas the eyes do not show signs of asymmetry at these stages. In both sinistral and dextral flounders, the Nodal-pathway genes are expressed at the left side of the dorsal diencephalon and left lateral-plate mesoderm. Considering these data together with our previous finding that reversal of eye laterality occurs to some extent in the P. olivaceus mutant reversed, in which embryonic pitx2 expression is randomized, we propose that although the Nodal pathway seems to function to fix eye laterality, embryonic expression of these genes does not act as a direct positional cue for eye laterality.
  ZOOLOGICAL SCIENCE 03/2007; 24(2):137-46. · 0.95 Impact Factor
• Article: Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning.

  Hisashi Hashimoto, Susumu Uji, Tadahide Kurokawa, Youhei Washio, Tohru Suzuki
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  ABSTRACT: The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.
  Gene 02/2007; 387(1-2):126-32. · 2.34 Impact Factor
• Article: Structural comparison of promoter and coding sequence of type I collagen alpha 1 chain gene duplicates between zebrafish and flounder/fugu lineages.

  Tohru Suzuki, Anand S Srivastava, Hisashi Hashimoto, Tadahide Kurokawa
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  ABSTRACT: Alpha 1 chain (Colalpha1(I)) and alpha 2 chain (Colalpha2(I)) are universal components of type I collagen in tetrapods, but rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) have a third: alpha 3 chain (Colalpha3(I)). This study tests whether Colalpha3(I) is a duplicate of Colalpha1(I) by whole-genome duplication (WGD) that occurred early in the ray-fin fish lineage. We also examine how their promoter sequence was modified after WGD. We cloned Colalpha1(I), Colalpha2(I) and Colalpha3(I) cDNAs and their promoters from flounder (Paralichthys olivaceus) and obtained corresponding sequences from the genome databanks of two pufferfishes Takifugu rubripes and Tetraodon nigroviridis, by BLAST-Search using flounder sequences. Phylogenetic analysis of N-terminal sequences of ca. 100 amino acids, including signal peptide and N-propeptide sequences before short triple helical domain, indicates that Colalpha3(I), found only in teleosts, is a duplicate of Colalpha1(1) by WGD. Colalpha1(I) and Colalpha3(I) genes begin to be transcribed at different stages of Takifugu embryogenesis, suggesting that their structure of promoter is modified differently after WGD. In flounder, Takifugu and Tetraodon, the structure of proximal region of promoter is highly conserved within Colalpha1(I) and within Colalpha3(I); no homology is apparent except for the TATA element motif between Colalpha1(I) and Colalpha3(I) of each species. Unexpectedly, zebrafish Colalpha1(I) promoter is more homologous to Colalpha3(I) of flounder and fugu than Colalpha1(I) is. These results suggest that each duplicated Colalpha1(I) gene promoter inherited a unique structure after WGD, but the manner of modification differed between the phylogenetically separated zebrafish and flounder/pufferfish lineages.
  Comparative Biochemistry and Physiology Part D Genomics and Proteomics 03/2006; 1(1):20-7. · 1.72 Impact Factor
• Article: Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes.

  Susumu Uji, Tohru Suzuki, Tadahide Kurokawa
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  ABSTRACT: Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.
  Comparative Biochemistry and Physiology Part D Genomics and Proteomics 03/2006; 1(1):133-8. · 1.72 Impact Factor
• Article: Identification of cDNA coding for a homologue to mammalian leptin from pufferfish, Takifugu rubripes.

  Tadahide Kurokawa, Susumu Uji, Tohru Suzuki
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  ABSTRACT: We identified cDNA coding for a homologue to mammalian leptin in puffer, Takifugu rubripes, using genomic synteny around the human leptin gene. In addition to significant sequence homologies, the puffer leptin (pLEP) displays characteristic structural features in common with mammalian leptin. The pLEP mRNA was expressed mostly in the liver that contained abundant lipids. In addition, homologues to pLEP were found in the databanks for three fish species (salmon, medaka, and Tetraodon) and two amphibians (salamander and Xenopus). The phylogenetic analysis shows rapid rates of molecular divergence among leptins from different vertebrate classes, but not between mammals and avians.
  Peptides 06/2005; 26(5):745-50. · 2.43 Impact Factor