Molecular and genetic studies suggest that thyroid hormone receptor is both necessary and sufficient to mediate the developmental effects of thyroid hormone

Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopuslaevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general.     

Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog

The current review focuses on the molecular mechanisms and developmental roles of thyroid hormone receptors (TRs) in gene regulation and metamorphosis in Xenopus laevis and discusses implications for TR function in vertebrate development and diversity. Questions addressed are: (1) what are the molecular mechanisms of gene regulation by TR, (2) what are the developmental roles of TR in mediating the thyroid hormone (TH) signal, (3) what are the roles of the different TR isoforms, and (4) how do changes in these molecular and developmental mechanisms affect evolution? Even though detailed knowledge of molecular mechanisms of TR-mediated gene regulation is available from in vitro studies, relatively little is known about how TR functions in development in vivo. Studies on TR function during frog metamorphosis are leading the way toward bridging the gap between in vitro and in vivo studies. In particular, a dual function model for the role of TR in metamorphosis has been proposed and investigated. In this model, TRs repress genes allowing tadpole growth in the absence of TH during premetamorphosis and activate genes important for metamorphosis when TH is present. Despite the lack of metamorphosis in most other vertebrates, TR has important functions in development across vertebrates. The underlying molecular mechanisms of TR in gene regulation are conserved through evolution, so other mechanisms involving TH-target genes and TH tissue-sensitivity and dependence underlie differences in role of TR across vertebrates. Continued analysis of molecular and developmental roles of TR in X. laevis will provide the basis for understanding how TR functions in gene regulation in vivo across vertebrates and how TR is involved in the generation of evolutionary diversity.     

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

We studied the profiles of 3,5,3'-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 TRbeta cDNA clone was generated by RACE. Two TRalpha 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.     

The thyroid gland and thyroid hormones in Senegalese sole (Solea senegalensis) during early development and metamorphosis

We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea senegalensis), and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Under our rearing conditions at 19.5 degrees C, most larvae entered metamorphosis in stage 1 at 15 days post-hatching (dph), and completed metamorphosis in stage 4 at 25dph. The onset of metamorphosis coincided with surges in whole body T4 and T3 concentrations. Crossmon's trichrome stain colored the lumen of follicular structures brightly red, and this co-localized with a T4-immunoreactivity. Thyroid follicles were first observed in stage 0 pre-metamorphic larvae at 5dph of age, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Increases in whole body thyroid hormone levels coincided with a 2(1/2)-fold increase in the total thyroidal colloid area in stage 1 larvae (aged 15dph) compared to stage 0 larvae (12dph). This was preceded by an approximately 40%-increase in the follicles' epithelial cell height in stage 0 larvae at 12dph compared to larvae at 5dph, and by an increase in the whole body T3/T4 ratio, indicative of an increase in outer ring deiodination. We conclude that in S. senegalensis there is a clear chronology in the activation of the thyroid gland that starts in early pre-metamorphic larvae.     

Amphibian metamorphosis as a model for studying endocrine disruption on vertebrate development: Effect of bisphenol A on thyroid hormone action

Thyroid hormone (TH) is essential for proper development in vertebrates. TH deficiency during gestation and early postnatal development produces severe neurological, skeletal, metabolism and growth abnormalities. It is therefore important to consider environmental chemicals that may interfere with TH signaling. Exposure to environmental contaminants that disrupt TH action may underlie the increasing incidence of human developmental disorders worldwide. One contaminant of concern is the xenoestrogen bisphenol A (BPA), a chemical widely used to manufacture polycarbonate plastics and epoxy resins. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. As TH action at the cellular level is highly conserved across vertebrate species, amphibian metamorphosis serves as an important TH-dependent in vivo vertebrate model for studying potential contributions of BPA toward human developmental disorders. Using Xenopus laevis as a model, we and others have demonstrated the inhibitory effects of BPA exposure on metamorphosis. Genome-wide gene expression analysis revealed that surprisingly, BPA primarily targets the TH-signaling pathway essential for metamorphosis in Xenopus laevis. Given the importance of the genomic effects of TH during metamorphosis and the conservation in its regulation in higher vertebrates, these observations suggest that the effect of BPA in human embryogenesis is through the inhibition of the TH pathway and warrants further investigation. Our findings further argue for the critical need to use in vivo animal models coupled with systematic molecular analysis to determine the developmental effects of endocrine disrupting compounds.     

Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis

Thyroid hormone (TH) induces the dramatic morphological and physiological changes that together comprise amphibian metamorphosis. TH-responsive tissues vary widely with developmental timing of TH-induced changes. How larval tadpole tissues are able to employ distinct metamorphic programs in a developmental stage- and TH-dependent manner is still unknown. Recently, several proteins capable of transporting TH have been identified. TH action and metabolism occurs primarily intracellularly, highlighting the importance of TH transporters. We examined the hypothesis that TH transporter expression and tissue distribution play an important role in mediating TH-induced metamorphic events. Xenopus tropicalis homologs for known TH transporting OATP, MCT and LAT family proteins were identified and gene specific qRT-PCR primers were developed. Total RNA was extracted from tissues representing three unique developmental fates including: growth/differentiation (hind limb), death/resorption (gill, tail) and remodeling (brain, liver, kidney). For growing and resorbing tissues, results showed the general trend of low initial expression levels of MCT8 and MCT10 transporters, followed by a several-fold increase of expression as the tissue undergoes TH-dependent metamorphic changes. The expression pattern in remodeling tissues was less uniform: a general decrease in transporter expression was observed in the liver, while the kidney and brain exhibited a range of expression patterns for several TH transporters. Collectively, these developmental expression patterns are consistent with TH transporting proteins playing a role in the effects of TH in peripheral tissues.     

Molecular cloning of prepro-thyrotropin-releasing hormone cDNA from medaka (Oryzias latipes)

The cDNA encoding prepro-thyrotropin-releasing hormone (ppTRH) in a teleost, medaka (Oryzias latipes) was isolated and characterized. The medaka ppTRH cDNA codes for 270 amino acid residues including eight TRH progenitor sequences (-Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg-). In silico analyses of the medaka genome database predicted that the structure of the medaka ppTRH gene is similar to the ppTRH genes of the other vertebrate species studied to date; consisting of three exons and two introns. Identity of the medaka ppTRH with the other vertebrates is rather low except the sockeye salmon. A molecular phylogenic tree showed that the ppTRH sequences reflected the predicted pattern of species classification. RT-PCR analysis demonstrated ppTRH gene expression in the brain and retina. These results gave some insight into the molecular evolution of ppTRH and physiological functions of TRH in vertebrates.     

Higher thyroid hormone receptor expression correlates with short larval periods in spadefoot toads and increases metamorphic rate

Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have (1) late onset of TR expression prior to the production of endogenous TH and (2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its higher TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.     

甲状腺功能减退对新生早期大鼠各脑区甲状腺激素受体mRNA表达的影响

目的探讨甲状腺功能减退（甲减）对新生早期大鼠各脑区甲状腺激素受体（TR）mRNA表达的影响。方法建立甲减Wistar大鼠动物模型，分别于仔鼠0、14、21、45d，用实时荧光定量PCR方法检测大脑、小脑、脑干和海马TR mRNA的表达。结果与对照组相比，各时间点甲减仔鼠各脑区TRα1 mRNA的表达量呈总体下调趋势，而甲减0d仔鼠大脑、小脑、脑干TRα2 mRNA表达量明显增高（t=8．18、6．23、3．68，P〈0．01），且45d仔鼠各脑区TRα2 mRNA表达量仍高于对照组（t大脑=5．50、t小脑=5．46、t脑干=4．10、t海马=11．83，P〈0．01），TRα1、TRα2 mRNA表达峰（21d）均延迟于对照组（14d）出现。甲减仔鼠TRβ1 mRNA表达变化趋势与对照组相一致，但45d仔鼠各脑区TRβ1 mRNA表达量均低于对照组（t大脑=4．64、t小脑=2．73、t脑干=3．90、t海马=5．07，P〈0．01或〈0．05）。结论甲减时甲状腺激素受体mRNA表达峰值的延迟出现以及异常的表达变化与克汀病脑损伤机制密切相关。     

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235 bp for gERα, 1967 bp for gERβ1 and 2158 bp for gERβ2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERβ1 mainly expressed in immature ovaries and gERβ2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERβ1 increased gradually, and gERβ2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.     

Characterization of thyroid hormone receptor alpha and beta in the metamorphosing Japanese conger eel,Conger myriaster

Two distinct TR alpha cDNA clones (TR alpha A and TR alpha B) were isolated from conger eel (Conger myriaster). The deduced amino acid sequences of the conger eel TR alphas showed higher homologies to the TR alphas of other vertebrates than to TR betas. Determination of TR mRNA in metamorphosing eels was performed using competitive RT-PCR. Of the two TR alpha mRNAs identified, TR alpha A mRNA expression was shown to be relatively higher than that of TR alpha B, and there was a peak in the expression of each during metamorphic climax. We hypothesize that both TR alphas play important roles in morphological differentiation during metamorphosis. The expression pattern of TR beta 1 mRNA was also higher during metamorphic climax and high levels of expression continued after metamorphosis. This suggests that TR beta 1 is the adult fish form which appears at high frequency after metamorphosis. It was also shown that TR beta 2 is highly expressed specifically in the brain and pituitary gland in larvae and juvenile forms including during metamorphosis and there was a peak in TR beta 2 mRNA in the elver after metamorphosis. Thus, we propose that TR beta 2 plays an important role in the regulation of the hypothalamic-pituitary-thyroid axis.     

Cloning of Atlantic halibut growth hormone receptor genes and quantitative gene expression during metamorphosis

To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5-10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (81%) and type II GHRs (58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis.