Characterization of thyroid hormones and thyroid hormone receptors during the early development of Pacific bluefin tuna (Thunnus orientalis)

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General and Comparative Endocrinology 155 (2008) 597–606 www.elsevier.com/locate/ygcen

Characterization of thyroid hormones and thyroid hormone receptors during the early development of Pacific bluefin tuna (Thunnus orientalis) Yutaka Kawakami b

a,*

, Jun Nozaki a, Manabu Seoka b, Hidemi Kumai b, Hiromi Ohta

a

a Department of Fisheries, Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan Uragami Experiment Station, Fisheries Laboratory, Kinki University, Nachikatsuura, 649-5145 Wakayama, Japan

Received 21 March 2007; revised 10 September 2007; accepted 11 September 2007 Available online 19 September 2007

Abstract We studied the profiles of 3,5,3 0 -L-triiodothyronine (T3), thyroxine (T4), and thyroid hormone receptors (TRs) in Pacific bluefin tuna (Thunnus orientalis) during embryonic and post-embryonic development. Both T3 and T4 were detected in embryos just before hatching, and it was found that the levels of both were increased in postflexion fish. The thyroid follicles were increased in both size and number in postflexion fish compared with preflexion fish. A TRb cDNA clone was generated by RACE. Two TRa cDNA clones were also partially identified and analyzed by real-time RT-PCR in this study. The TR mRNA levels in embryos were determined, and these were found to be lower than those in preflexion fish. Therefore, we considered that thyroid hormones function during early post-embryonic development as well as during embryonic development. Moreover, there was a peak in the TR mRNA level during postflexion stages, as seen during metamorphosis in Japanese flounder and Japanese conger eel. It is possible that thyroid hormones control the early development of scombrid fish through TRs, as they do for Pluronectiformes and Anguilliformes. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Early development; T3; T4; Thyroid hormone receptor; Pacific bluefin tuna

1. Introduction Metamorphosis takes place in certain species of fish in association with a transition to a new habitat. This type of metamorphosis involves an ontogenetic transformation from a larval form to a juvenile one (Wilbur, 1980; Youson, 1988), and is termed first (true) metamorphosis in contrast with second metamorphosis, which is restricted to events related to final sexual maturation and involves the full expression of adult genes involved in reproduction (Youson, 1997). In particular, the morphological changes that occur in eels (Anguilliformes: development from leptocephalus to elver; Smith, 1979) and flounder (Pleuronectiformes: eye migration; Keefe and Able, 1993; Minami, 1982) are representative examples of metamorphosis (Wilbur and Collins, 1973). *

Corresponding author. Fax: +81 743 75 5966. E-mail address: [email protected] (Y. Kawakami).

0016-6480/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.09.005

Metamorphosis in these species is under the influence of thyroid hormones (THs) (Anguilliformes: Kitajima et al., 1967; Pleuronectiformes: Inui et al., 1994; Schreiber and Specker, 1998; Solbakken et al., 1999). Moreover, the expression patterns of the TH receptor (TR) and the relationships between THs and TR mRNA levels have been studied in Pleureonectiformes (Yamano and Miwa, 1998) and Anguilliformes metamorphosis (Kawakami et al., 2003a, b). Youson (1997) suggested that metamorphosis is a term that has been used rather loosely by fish biologists to describe both major and minor transformations of larval phenotypic characters into those of adults. Like the major transformations influenced by THs seen in Anguilliformes and Pleureonectiformes, the minor transformations seen in species such as black seabream (Perciformes: Acanthopagrus schlegelii; Tanaka et al., 1991), red seabream (Perciformes: Pagrus major; Hirata et al., 1989),

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and coral trout grouper (Perciformes: Plectropomus leopardus; Trijuno et al., 2002) are also indicated to be under the influence of THs. It is possible that THs play roles in the early development of many fish. Morphological characteristics common to the early development of scombrid fish include a large head, gape and eyes, development of head spination, and posterior migration of the anus (Collette et al., 1984). However, the role of THs in the early development of scombrid fish is not understood at all. Characterizing the role of TRs in the early development of scombrid fish will increase our understanding of the roles of THs in types of early developments other than those utilized by Pleuroeonectiformes and Anguilliformes. In this study, we use the early development of fish and juveniles of Pacific bluefin tuna (Perciformes, Thunnus orientalis) as a model for the early development of scombrid fishes, and we investigate the whole body TH and TR mRNA transcript levels from embryos to juveniles. In addition, we compare the expression of TRs during early development in Pacific bluefin tuna with previously published data on the Japanese flounder (Pleureonectiformes: Paralichthys olivaceus) and the Japanese conger eel (Anguilliformes: Conger myriaster). 2. Materials and methods 2.1. Animals Immature 1-year-old Pacific bluefin tuna (10–15 kg body weight), cultivated in a net cage at the Kinki University Fish Nursery Center at Amami in Kagoshima Prefecture, were collected in July 2004. Liver samples from Pacific bluefin tuna were frozen in liquid nitrogen and stored at 80 °C until required.

Fertilized Pacific bluefin tuna eggs, which had been naturally spawned in the Kinki University Fish Nursery Center at Amami, were obtained in July 2006 and transferred to 500-L round plastic and 40-ton round concrete tanks in the Kinki University Fish Nursery Center at Uragami in Wakayama Prefecture. At the time embryos were transferred to the rearing tank, those corresponding to the tail-bud stage were sampled, frozen in liquid nitrogen and stored at 80 °C until required. The water temperature in the 500-L round plastic tank ranged from 25.2 to 28.3 °C, while that in the 40-ton round concrete tank was around 27 °C. The feeding scheme for fish in either tank was as follows: rotifers, Branchionus rotundiformis, from 1 to 15 days post hatching (dph), Artemia nauplii, from 10 to 21 dph, and other live fish (Oplegnathus fasciatus) from 15 to 25 dph. Pacific bluefin tuna were sampled every day for 25 days, except on day 24 post hatching, from the 500-L round plastic tank. Fish were fixed with 5% neutral buffered formalin in 47.5% distilled water and 47.5% seawater or frozen in liquid nitrogen, and stored at 80 °C until required. In this study, morphological development from post-embryo to juvenile, including transformation, was separated into 17 stages, as shown in Fig. 1 and Table 1. In this study, specimens at stage Q were not sampled during the breeding period.

2.2. Reverse transcription (RT)-PCR and cDNA cloning Total RNA was extracted from Pacific bluefin tuna livers using Trizol Reagent (Invitrogen, Carlsbad, CA). Poly(A) + RNA was subsequently isolated from total RNA using Oligotex-dt-30 (Takara, Otsu, Japan). Isolated RNA was denatured at 70 °C for 10 min, placed on ice, and reverse-transcribed with Superscript II (Invitrogen) at 42 °C for 40 min, and at 50 °C for 30 min, using oligo(dT)12–18 as the anchor primer. For the amplification of Pacific bluefin tuna TRb (bTRb) cDNA fragments and TRa (bTRa) cDNA fragments, sense (bTRb: bTR-bDP-S; bTRa: bTR-DP-S), and antisense degenerate primers (bTRb: bTRbDP-A; bTRa: bTR-DP-A) were designed from the consensus sequence of an alignment of the deduced amino acid sequences of TRs from several vertebrate species, using DNASIS version 3.5 (Hitachi Software Engineering, Yokohama, Japan) (Table 2). PCR was carried out in a final volume of 50 ll containing 0.5–1 pg of cDNA, 400 nM primers, 800 lM dNTPs, and 2.5 U of Ex Taq (Takara). PCR was carried out

Fig. 1. Developmental stages of hatchery-reared Pacific bluefin tuna.

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Table 1 Characterization of stages according to Fig. 1 of hatchery-reared Pacific bluefin tuna Stage

Characterization

Ranges of TL (mm)

Ranges of dph

A B C D E F G H I J K L M

Hatched yolk-sac larvae The yolk-sac dwindles and its surface is covered in dendritic melanophores The mouth opens and the intestine starts to become coiled The yolk-sac disappears The intestine becomes coiled. Some specimens have a few spines in the preopercle Teeth appear. All specimens at this stage have a few spines Notochord flexion begins A few fin rays appear in the first dorsal fin Melanophores appear in the first dorsal fin The melanophores of the first dorsal fin develop. Notocord flexion progresses Second fin rays appear. Notochord flexion ends Postflexion larvae. Pelvic fin ray development is completed and the nasal cavity is separated completely Postflexion larvae. Pectoral fin rays appear during stages L and M. The number of rays in the dorsal, anal, and caudal fins increases The number of rays in the dorsal, anal, and caudal fins is complete Early juvenile stage. Pectoral fin rays are complete. Shape of rostral end get rounded. Ossification is started Middle juvenile stage. The preopercle spines begin to regress; they disappear during this stage. In the opercle, a thread of melanophores is observed Late juvenile stage. The specimens are heavily pigmented (from seven to 12 spots), with characteristic lateral blotches and numerous melanophores around the head. The first and second dorsal fins are completely separated, as are each of the finlets

2.7–3.0 3.6–3.7 3.5–3.8 3.5–3.8 3.4–4.4 4.3–5.3 4.5–6.4 6.4–6.6 6.5–7.5 7.0–7.6 7.5–10.1 9.2–12.0 10.0–13.5

1 2–3 3–4 4–8 7–10 9–12 10–11 10–14 11–14 12–16 14–16 14–20

12.2–17.0 16.2–33.8 21.3–41.0

17–21 22–25 Around 25

N O P Q

Over 40.0

dph, days post hatching.

Table 2 Primers used for cloning, PCR and real-time RT-PCR analysis of Pacific bluefin tuna TRs (bTRs) Name

Primer sequence

bTR-DP-S bTR-DP-A bTRbDP-S bTRbDP-A bTRb-UTR-S bTRb-UTR-A bTRaA-ST-T7-S

5 0 -WRMTGCATCATCGACAARRTSACC-3 0 5 0 -TGGGDCACTCSACYTTCATGTG-3 0 5 0 -WGRWAKTACCCAGATGTCTGAGG-3 0 5 0 -GCAGGCGGCCGCGAATTCACTAKTG-3 0 5 0 -TCTTGGCCTTGAACCCCACCAGT-3 0 5 0 -GCAGGCGGCCGCGAATTCACTAGT-3 0 5 0 -TGTAATACGACTCACTATAGGGTAAAA AGTGCATCGCTGTGGGG-3 0 5 0 -GACAACGCGTGTGATGGCGGGG-3 0 5 0 -TGTAATACGACTCACTATAGGGTATTGC AGTGGGCATGGCCATG-3 0 5 0 -AACGACACGGGTGATGGCAGGA-3 0 5 0 -TGTAATACGACTCACTATAGGGAGCAA GAGGCTGGCCAAGCGGAAG-3 0 5 0 -CACCACTCGGGTTATGGCAGGG-3 0 5 0 -TCAGATGATATTGGGCAGGGTC-3 0 5 0 -GACAACGCGTGTGATGGCGG-3 0 5 0 -AAGAAAACAGGGAGCGACGC-3 0 5 0 -ATGGGCTTCCGTCACCAGGC-3 0 5 0 -AACTGCAGAAGACGGCATGG-3 0 5 0 -TTCGCCCCCGCTGCACTCA-3 0

bTRaA-ST-A bTRaB-ST-T7-S bTRaB-ST-A bTRb-ST-T7-S bTRb-ST-A bTRaA-S bTRaA-A bTRaB-S bTRaB-A bTRb-S bTRb-A

Nucleotide numbers corresponding to the annealing site (Fig. 2)

1–23 bp 1220–1243 bp

406–425 bp 535–553 bp

bTR-DPs (degenerate primers), primers for amplification of bTR cDNA fragments; bTRb-UTRs (untranslated regions), sense and antisense primers for the sequencing of bTRb containing the open reading frame; bTR-ST (standard)-T7s and bTR-STs, sense and antisense primers constructing fragments for standard real-time RT-PCR analysis of bTRs; bTRs, sense and antisense primers for real-time RT-PCR analysis of bTRs.

for 35 cycles using a Thermal Cycler Dice Gradient (Takara) under the following conditions: denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. PCR products were separated by 1% agarose gel electrophoresis, and selected bands were cut out and purified using a QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands). Purified DNA fragments were subcloned into the plasmid vector pGEM-T Easy (Promega, Madison, WI) using a Ligation-Conve-

nience Kit (Nippon Gene, Tokyo, Japan), and positive clones were sequenced using a CEQ DTCS-Quick Start Kit (Beckman Coulter, Fullerton, CA), and a CEQä 8800 Genetic Analysis System (Beckman Coulter). bTRa cDNA products representing independent PCR subclones were each sequenced 5 times to account for errors acquired during PCR. Two bTRas were named using phylogenetic tree analysis based on a comparison with Japanese flounder TRas.

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Fig. 2. Nucleotide sequence of Pacific bluefin tuna thyroid hormone receptor b (bTRb) with the predicted amino acid sequence given below. Putative zinc fingers are indicated by a dotted line beneath the sequence. Sense (bTRb-UTR-S) and antisense (bTRb-UTR-A) primers were also designed against the untranslated regions of bTRb, based on primers designed against the untranslated regions of the TRbs of other teleostean

fish (Table 2). PCR was carried out as described above (denaturation at 94 °C for 30 s, annealing at 66 °C for 30 s, and extension at 72 °C for 30 s). The resulting PCR products were sequenced following the method

Y. Kawakami et al. / General and Comparative Endocrinology 155 (2008) 597–606 described above and bTRb cDNAs containing the entire open reading frame were obtained. bTRb cDNA products representing independent, full-length PCR clones were each sequenced 5 times to account for errors acquired during PCR.

2.3. Sequence analysis The deduced amino acid sequence, molecular weight and other traits of the proteins encoded by the TR cDNAs were determined using DNAsis software (Hitachi Software Engineering, Tokyo, Japan). GenBank Accession Numbers for the following TR genes were retrieved: Atlantic halibut (Hippoglossus hippoglossus) TRa (AF14396); Atlantic salmon (Salmo salar) TRa (AF146775); gilthead seabream (Sparus aurata) TRb1 (AY246695); Japanese conger eel TRaA, -aB, -bA(b1), and -bB(b2) (AB183396, AB183397, AB183394, and AB183395, respectively); Japanese flounder TRaA, -aB, and b1 (D16461, D16462, and D45245, respectively); Pacific bluefin tuna TRaA, -aB, and -b (AB332044, AB332045, and AB332043, respectively); zebrafish (Danio rerio) TRa1 and -b1 (U54796 and AF109732, respectively). A phylogenetic tree was constructed by neighbor-joining methods using CLUSTL W version 1.8 software (Bioinfomatics, Cent. Ins. Chem. Res. Kyoto Univ.), by running 1000 bootstrap replicates.

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We prepared sense primers against the T7 promoter site, and antisense primers, and made appropriately sized bTR cDNA fragments using PCR (Table 2). PCR fragments for standard template synthesis were purified by phenol/chloroform/isoamylalcohol extraction, and ethanol precipitated. cRNA standards were synthesized from 100 ng of PCR fragments, a mixture of NTPs and T7 RNA polymerase using a SP6/T7 transcription Kit (Roche). DNase I was added to the reaction mixtures for 30 min at 37 °C, and cRNAs were purified in phenol and ethanol precipitated. Purified cRNAs were quantified by UV260 absorbance and a 1/10 dilution series was constructed (106–101 copies/ll) using EASY Dilution (Takara). Each dilution series was reverse-transcribed as described above. RT-PCRs and a dilution series of standard samples were prepared according to the manufacturer’s protocol to include: 1 ll of cDNA, 10 ll of iQ SYBR Green Supermix (BioRad) and 100 nM primers in a final volume of 20 ll. DNA amplifications were performed in duplicate under the following conditions: 3 min at 95 °C, followed by 40 cycles of 10 s of denaturation at 95 °C, 20 s of annealing at 65 °C, and 20 s of extension at 72 °C. Standard templates were used, in duplicate, to construct a standard curve ranging from 101 to 106 copies. The linear range of the curves fell within 101– 106 copies and correlation coefficients were greater than 0.997 for all curves. The intra-assay coefficients of variation were 0.4–1.7% for bTRaA, 0.1–1.2% for bTRaB and 0.2–1.6% for bTRb. Inter-assay coefficients of variation were under 10.0% for bTRs, respectively.

2.4. Histological procedure Whole bodies of larvae fixed in 5% neutral buffered formalin were embedded in paraffin and sectioned at 7–10 lm. All sections were cut in parallel through a plane including the eyes. The sections were deparaffinized, rehydrated and stained with hematoxylin–eosin (HE).

2.5. Extraction and enzyme immunoassay of thyroid hormones The procedures for the extraction of T4 and T3 from samples followed those of Tagawa and Hirano (1990). Individual frozen pooled samples comprising 50 embryos, 30 fish of 3 dph and 30 of 5 dph, 10 fish of 10 dph, 5 fish for each of 13, 15, 18, and 20 dph, or one fish of 23 dph, were homogenized in 1 ml of ice-cold methanol. After centrifugation, the supernatants were saved, and 1 ml of methanol was added to each pellet, which was rehomogenized and recentrifuged. Total methanol extracts were dried out and reconstituted with 100 ll of methanol, 400 ll of chloroform and 100 ll of barbital buffer. After vortexing and centrifugation, the upper layers were isolated. This step was performed twice. Total extracts were dried out and this residue was reconstituted with 100 ll of barbital buffer. T3 and T4 enzyme immunoassay tests (ClinPro, Union City, CA) were performed in duplicate using up to 50 ll of each sample. Standard templates were used, also in duplicate, to construct a standard curve ranging from 0.9 to 19.0 pg/ml for T3 and from 0.3 to 7.0 lg/ml for T4. The correlation coefficients in the linear range of the curves were greater than 0.980 for all curves. The intra-assay coefficients of variation were 1.9–9.7% for T3 and 4.1– 12.1% for T4.

2.6. Real-time RT-PCR Real-time RT-PCR analyses were performed using a MiniOpticon Real-Time Detection System (BioRad). Specific primers for bTRs were designed based on the cDNA nucleotide sequences analyzed in this study (Table 1), using Primer Express Software version 1.0 (Applied Biosystems, Foster City, CA). Total RNA extracted from 30 to 50 embryos/fish in the case of stages between embryo and stage G, and from one fish in the case of stages between stages I–P, was extracted using Trizol Reagent (Invitrogen). Synthesis of first-strand cDNA was carried out as follows. One microgram of isolated total RNA was denatured at 70 °C for 5 min, immediately put on ice, and then reverse-transcribed by 100 U of M-MLV (Invitrogen) at 42 °C for 60 min, using 10 lM random primers as the cDNA synthesis primers.

3. Results 3.1. Nucleotide and predicted amino acid sequences of bTR cDNAs The nucleotide sequence of bTRb is shown in Fig. 2. The cDNA encoding bTRb contains an open reading frame of 1185 bp, encoding a protein of 395 amino acids. This protein contains two putative cysteine-rich zinc fingers (underlined in Fig. 2), which are characteristic of all nuclear hormone receptors, including TRs. In order to isolate a region of sequence for bTRas, we designed a series of degenerate PCR primers. For each Pacific bluefin tuna RTPCR, fragments for TRa were obtained at the expected size. From each subcloned PCR fragment, three clones were selected for sequencing. The resulting sequences were aligned with known TRs from teleostean fish to check their authenticity (Fig. 3). bTRas are more closely related to the TRas of other teleostean fish than bTRbs are to the TRbs of other teleostean fish. A phylogenetic analysis of teleostean fish TR sequences is shown in Fig. 4. TRa and TRb sequences form two separate clusters. bTRaA and bTRaB sequences are most similar to the TRaA and TRaB sequences of Japanese flounder. 3.2. Appearance of thyroid gland and expression of THs from embryonic to juvenile stages The thyroid follicles had a narrow perimeter of epithelial cells surrounding the colloid and lumen at stage E (5 dph; arrow in Fig. 5E). The thyroid follicles increased in both size and number during early development (Fig. 5). Fig. 6 shows the changes in T4 and T3 concentrations between the embryonic stage and stage P (23 dph). THs were present at low levels in embryos. TH concentrations were maintained at low levels after hatching, whereas, in

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Fig. 3. Comparison of the amino acid sequences of thyroid hormone receptors (TRs) from the Japanese eel (Anguilla japonica), Japanese conger eel (Conger myriaster), Japanese flounder (Paralichthys olivaceus), Atlantic halibut (Hippoglossus hippoglossus), gilthead seabream (Sparus aurata), Pacific bluefin tuna (Thunnus orientalis), zebrafish (Danio rerio), and Atlantic salmon (Salmo salar). Vertical lines indicate the borders of four domains: the Nterminal hypervariable region (A/B domain), the DNA-binding domain (C domain), a hinge region (D domain), and the ligand-binding domain (E/F domain).

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Fig. 4. Phylogenetic tree of 15 vertebrate thyroid hormone receptors (TRs). The lengths of horizontal lines indicate genetic distance. One thousand bootstrap replicates were performed; values are shown at the inner nodes.

Fig. 5. Sagittal section through the subpharyngeal region of a Pacific bluefin tuna, parallel with a plane including the eye, stained with HE. E, subpharyngeal region of a fish at 5 dph (stage E according to Fig. 1). H, subpharyngeal region of a fish at 11 dph (stage H according to Fig. 1). O, subpharyngeal region of a fish at 23 dph (O stage according to Fig. 1). Arrows in E, H, and O indicate thyroid follicles.

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postflexion stage TH concentrations were elevated, reaching a peak. The concentration of T3 was lower than that of T4. 3.3. Changes in bTR mRNA levels from embryonic to juvenile stages Fig. 8 shows the changes in the expression levels of bTR mRNAs at different developmental stages, from embryos to stage P, as measured by real-time RT-PCR. The expression levels of bTRa mRNAs were relatively higher than those of bTRb mRNAs. All three bTRs were elevated in postflexion fish (M/N in Fig. 7). The bTRaA mRNA level peaked in preflexion fish and decreased in flexion fish. As well as the peaks in bTRaB and bTRb mRNA levels observed in postflexion fish, there was also a peak in the bTRaA mRNA level in postflexion fish. 4. Discussion

Fig. 6. Changes in TH content during the development of Pacific bluefin tuna. Values represent the means ± SEM of three independent pooled samples.

Hormones, including thyroid hormones, have been shown to pass into fish oocytes from the maternal circulation (Greenblatt et al., 1989). When TH concentrations were analyzed in unfertilized, fertilized and/or ovarian eggs just before spawning, from 26 teleostean species, THs were found in the eggs of all species (Tagawa et al., 1990). In

Fig. 7. Developmental changes in the expression levels of bTR genes. Black circles, the mean values of three independent pooled samples. White circles, the maximal values; gray circles, the minimum values.

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zebrafish (Danio rerio), cotreatment of embryos and fish with an antagonist of TR and an inhibitor of thyroid hormogenesis led to severe retardation of development, and TR mRNAs are expressed during embryonic and postembryonic development. From these results in zebrafish, Liu and Chan (2002) concluded that THs are important for embryonic to post-embryonic development. THs were detected in Pacific bluefin tuna just before hatching (Fig. 7); however, the expression patterns of THs in the unfertilized eggs of Pacific bluefin tuna were not analyzed in this study. Moreover, expression of bTR mRNAs was observed in the embryos of Pacific bluefin tuna (Fig. 7), and it may be that THs function in the embryonic development of Pacific bluefin tuna. We did not observe cells expressing TH in larvae of 1 dph by histological analysis (data not shown) and consider embryos not to have the capacity for thyroid hormonegenesis. We suggest that THs pass into eggs from the maternal circulation before ovulation, and function in embryonic development. Thyroid follicles are found at stage E (5 dph) (Fig. 5E). Moreover, the expression levels of bTR mRNAs during the preflexion stages are also higher than those in embryos (Fig. 7). Therefore, we considered that TH functions during early post-embryonic development as well as during embryonic development. In particular, the bTRaA mRNA level has a peak during stage G (Fig. 7). TH levels, especially the T4 concentration, were found to suddenly increase between 5 dph (E stage) and 10 dph (G/H/I stages); thus, it might be that TH functions mostly through bTRaA at these stages. In Pacific bluefin tuna, there was a peak in the levels of the three bTR mRNAs during the postflexion stages (Fig. 8). It is possible that THs control the early development of scombrid fish through bTRs, as they do in Pluronectiformes (Japanese flounder; Yamano and Miwa, 1998) and Anguilliformes (Japanese conger eel; Kawakami et al., 2003b). Fig. 8 summarizes the changes in the levels of TR mRNAs during the early development of Pacific bluefin tuna, Japanese flounder (modified from Yamano and Miwa, 1998) and Japanese conger eel (Kawakami et al., 2003b). During the metamorphosis of the latter two teleostean fish and during the early development of Pacfic bluefin tuna, the expression levels of TRa mRNAs have distinct peaks; thus, it might be possible to consider the expression patterns of the TRas of these three teleostean fish in the same way. Regarding TRbs, the levels of Japanese flounder TRb and Japanese conger eel TRbA mRNAs increase during metamorphosis, and a high level of expression is maintained. We proposed that Japanese conger eel TRbA is the adult type, and that its levels remain elevated after metamorphosis (Kawakami et al., 2003b). Japanese conger eel and Japanese eel (Anguilla japonica) TRbBs are highly expressed in the brain and pituitary (Kawakami et al., 2003b, 2007). It is not known whether high expression of a brain and pituitary type of TRb occurs in other teleostean fish, including Pacific bluefin tuna. We considered the possibility that a highly expressed brain and pituitary

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Fig. 8. Developmental changes in the expression of TR genes during transformation. (a) bTR genes analyzed in this study. (b) Japanese flounder TR genes modified from Yamano and Miwa (1998). (c) Japanese conger eel TR genes in Kawakami et al. (2003b).

type of TRb might only be seen in Anguilliformes and/or lower fish. On the other hand, the level of bTRb mRNA showed a small peak in postflexion fish in the present study. Moreover, the expression levels of Japanese flounder TRb and Japanese conger eel TRbA mRNAs are about the same or higher than those of TRas; however, the expression level of bTRb mRNA is lower than those of bTRas. The difference in the expression levels of TRb mRNAs might be reflected in the differences in their roles during early development. It is now known what TRa and TRb genes control in teleostean fish; the next step will be to characterize the roles of TRs in each tissue, including during early development. References Collette, B.B., Potthoff, T., Richards, W.J., Ueyanagi, S., Russo, J.L., Nishikawa, Y. (1984). Scombroidei: development and relationships. In: Moser, H.G., Richards, W.J., Cohen, D.M., Fahay, M.P., Kendall, A.W., Jr., Richardson, S.L. (Eds.), Ontogeny and Systematics of

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