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Originally published In Press as doi:10.1074/jbc.M402847200 on March 30, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26555-26562, June 18, 2004
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Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1*

J. David Furlow{ddagger}§, Ha Yung Yang{ddagger}, Mei Hsu{ddagger}, Wayland Lim{ddagger}||, Davy J. Ermio{ddagger}, Grazia Chiellini**, and Thomas S. Scanlan**

From the {ddagger}Section of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616-8519 and the **Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0446

Received for publication, March 15, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major challenge in understanding nuclear hormone receptor function is to determine how the same ligand can cause very different tissue-specific responses. Tissue specificity may result from the presence of more than one receptor subtype arising from multiple receptor genes or alternative splicing. Recently, high affinity analogs of nuclear receptor ligands have been synthesized that show subtype selectivity. These analogs can greatly facilitate the study of receptor subtype-specific functions in organisms where mutational analysis is problematic or where it is desirable for receptors to be expressed in their normal physiological contexts. We describe here the effects of the synthetic thyroid hormone analog GC-1 on the metamorphosis of the frog Xenopus laevis. The most potent natural thyroid hormone, 3,5,3'-triidothyronine or T3, shows similar binding affinity and transactivation dose-response curves for both thyroid hormone receptor isotypes, designated TR{alpha} and TR{beta}. GC-1, however, binds to and activates TR{beta} at least an order of magnitude better than it does TR{alpha}. GC-1 efficiently induces death and resorption of premetamorphic tadpole tissues such as the gills and the tail, two tissues that strongly induce thyroid hormone receptor {beta} during metamorphosis. GC-1 has less effect on the growth of adult tissues such as the hindlimbs, which express high TR{alpha} levels. The effectiveness of GC-1 in inducing tail resorption and tail gene expression correlates with increasing TR{beta} levels. These results illustrate the utility of subtype selective ligands as probes of nuclear receptor function in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear hormone receptors and their ligands are essential regulators of metazoan development, reproduction, and homeostasis. One of the most dramatic nuclear receptor-mediated events in nature is amphibian metamorphosis (1, 2). In the frog, metamorphosis occurs as a result of gene expression cascades induced by thyroid hormone (TH)1 secreted from the developing tadpole's thyroid gland (3). These cascades result in dramatically different morphological responses such as the growth and differentiation of the limbs, the death and resorption of the larval gills and tail, and the remodeling of a large number of larval organs for new adult functions (1). As in all vertebrates, TH exerts its effects via a pair of TH receptor subtypes encoded by separate genes, TR{alpha} and TR{beta} (4). TRs are ligand-regulated transcription factors that are thought to repress target gene transcription in the absence of hormone and activate transcription upon hormone binding (5). The most detailed molecular studies on amphibian metamorphosis have been done on the frog Xenopus laevis. Xenopus TR{alpha} is expressed early in development (6, 7), long before the embryo and larval tadpole have a functional thyroid gland. Just prior to metamorphosis, xTR{alpha} is ubiquitously expressed, but particularly high levels of it are detected in the brain, limb buds, skin, and other tissues that are destined to respond to TH by proliferating and differentiating into adult organs (8, 9). In contrast, much lower levels of Xenopus TR{beta} mRNA are detectable prior to metamorphosis (6, 10). At metamorphosis, TR{beta} is strongly induced by TH in larval tissues such as the tail that will die and resorb. TR{beta} levels remain very low in the growing limbs, although expression in this tissue may be highly localized (11). In remodeling tissues, such as the intestine and cartilage of the head, TR{beta} mRNA is up-regulated in both dying and proliferating cells, suggesting a possible role for TR{beta} in both processes (8, 12, 13).

Xenopus TRs are extremely well conserved in comparison with their avian and mammalian counterparts (4). xTR{alpha} is >95 and 91% conserved in its DNA and ligand-binding domains, respectively, when compared with rat TR{alpha}. xTR{beta} is >96 and 94% conserved in those domains compared with rat TR{beta}. The major difference between the two Xenopus TR subtypes is the lack of an amino-terminal domain in xTR{beta} that may participate in cell-specific transcriptional activation or altered DNA binding properties in xTR{alpha}. A recently developed synthetic thyroid hormone analog, GC-1 (Fig. 1, bottom), preferentially binds and transactivates mammalian TR{beta} versus TR{alpha} (14). In human TRs, a single amino acid difference in the ligand binding pocket is responsible for this selectivity (15), and this amino acid difference is conserved in Xenopus TRs. In vivo, GC-1 selectively decreases plasma lipids and thyroid-stimulating hormone secretions without increasing the heart rate in hypothyroid rats (16). The effects of GC-1 are consistent with the known relative expression patterns of rat TR subtypes in the liver, the pituitary, and the heart. Recently, we have established that X. laevis metamorphosis provides a simple screening assay for the biological activity of thyroid hormone receptor antagonists (17). Because the Xenopus and mammalian TRs are strongly conserved and the tissue-specific responses to TH are very dramatic, we used GC-1 to probe TR subtype function in living X. laevis tadpoles during induced metamorphosis.



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  		FIG. 1.
Structures of T3 and GC-1. Chemical structures of T3 and the isotype selective agonist GC-1.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protease Protection Assays—[35S]methionine-labeled (Amersham Biosciences) TR{alpha} and TR{beta} proteins were synthesized using the TNT SP6-coupled wheat germ extract in vitro transcription and translation system (Promega). 2 µl of translated 35S-labeled TR was mixed with the indicated concentrations of T3, GC-1, NH-3, or vehicle and incubated for 30 min at room temperature. 2 µg of type IV elastase (Sigma) was mixed with the TR and hormone in 96-well plates (Costar 9017), bringing the total volume to 20 µl. After 20 min of incubation at room temperature, SDS-PAGE sample buffer was added followed by four 1-min freezethaw cycles. Samples were boiled for 10 min before SDS-PAGE analysis. The SDS-PAGE gel was fixed with 25% isopropanol and 10% acetic acid and dried for 2 h. The gel was exposed to a PhosphorImager screen overnight and scanned with Storm 860 (Amersham Biosciences). The bands were quantitated using ImageQuant software.

Transient Transfection Assays—DNA encoding the Xenopus TR{alpha} ligand-binding domain (amino acids 132–418) or Xenopus TR{beta} ligand-binding domain (amino acids 85–369) was amplified by PCR using proofreading (Pfu) DNA polymerase from cDNA encoding X. laevis TR{alpha} or TR{beta} using specific primers flanked by BamHI sites (5' and 3'). These fragments were subcloned in-frame with the Gal4 DNA-binding domain into pSG5 Gal4 DBD vector (a gift from Marty Privalsky, University of California, Davis) between the BamHI and BglII sites of the dephosphorylated vector. All clones were verified by DNA sequencing (University of California, Davis DNA Sequencing Facility). All cell culture media and transfection reagents were obtained from Invitrogen unless indicated otherwise. XLA kidney cells or XTC fibroblast cells were seeded in 6-well tissue culture plates at a density of 1 x 105 cells per well in 70% Leibovitz L-15 medium plus 10% fetal bovine serum and gentamycin. The cells were maintained at room temperature without CO2 supplementation. XLA cells were transfected with 50 ng of pSG5 Gal4/TRa or pSG5 Gal4/TRb, 100 ng of MH100X4 (a luciferase reporter gene with four upstream activation stream or Gal4 binding sites, kindly provided by David Mangelsdorf, University of Texas Southwestern), and 350 ng of pCS2-{beta}galactosidase per well (18). XTC cells were transfected with 100 ng of X. laevis TR expression vector (miw TR{alpha}, miw TR{beta}, or miw chloramphenicol acetyltransferase as control (19)), 100 ng of TH/bZIP TH response element-luciferase reporter vector (19), and 300 ng of pCS2-{beta}galactosidase (18). DNA for XLA or XTC cell transfections were mixed with 2 µl of LipofectAMINE 2000 (Invitrogen) per well and added to the cells for 24 h. Cells were allowed to recover for 12–16 h in the culture medium and then treated for 48 h with the indicated doses of T3, GC-1, or vehicle. Cells were harvested by scraping in 10 mM Tris-Cl, pH 7.8, 150 mM NaCl, and 1 mM EDTA, and cell pellets were resuspended in 125 µl of reporter lysis buffer (Promega) followed by a single freeze-thaw cycle. Cell extracts were assayed for luciferase and {beta}-galactosidase activity as described elsewhere (19).

Animal Studies—X. laevis tadpoles were purchased from Nasco, Inc. (Fort Atkinson, WI) and staged according to Nieuwkoop and Faber (20). All chemicals were purchased from Sigma unless indicated otherwise. For hormone treatments, a 1 mM stock of T3 was dissolved in 4 mM NaOH, and a 10 mM GC-1 stock was made in dimethyl sulfoxide. GC-1 was synthesized as described elsewhere (14). For whole animal treatments, stage 52 and stage 53 tadpoles were placed in 1x Steinberg's solution (10 mM HEPES, 60 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4, pH 7.4) with 1 mM methimazole and the indicated hormone concentrations or appropriate vehicle and observed daily. After 1 week, animals were fixed in phosphate-buffered saline containing 10% formalin. Animals were photographed with a Nikon F-5 camera, and slides were scanned into Adobe Photoshop 5.0 using a Kodak RFS 3570 Plus film scanner. Images were then assembled in Adobe Pagemaker 6.5.

Tail Organ Culture—Tadpole tails were cultured in vitro as described previously (21), with some modifications. Staged tadpoles were pre-treated in 1x Steinberg's solution containing gentamycin (70 µg/ml) and streptomycin (200 µg/ml) 24 h before removal of the tail tips for culture. The tadpoles were anesthetized briefly in water containing 0.01% MS222 (aminoesterbenzoic acid; Sigma), after which 6 mm of the tail tip was cut and placed in 1 ml of Steinberg's solution with antibiotics and 5 µg/ml insulin in 12-well Falcon tissue culture dishes (BD Biosciences). Tail tips were left in the culture media for 24 h before the first hormone treatment. T3 (Sigma) and GC-1 were diluted to the appropriate final concentration. The same final dilution of the vehicle was added to control cultures. Afterward, the culture media with hormones were changed every 48 h. The length of each tail was measured every 24 h, and each experimental point consisted of the mean change of 4 to 6 tails. Each experiment was repeated twice.

RNA Analysis—Tadpoles of the indicated stage were treated with T3 or GC-1 in 1x Steinberg's solution for 48 h. Tails were harvested and frozen in liquid nitrogen. Total RNA was isolated and analyzed by Northern hybridization using cDNA probes as described previously (22). The amount of hybridized probe was determined using an Amersham Biosciences Storm PhosphorImager and ImageQuant software and normalized to the expression of the ribosomal protein rpL8 gene (23).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GC-1 Binds Preferentially to X. laevis TR{beta} over TR{alpha}—GC-1 is an iodine-free thyromimetic that preferentially binds mammalian TR{beta} over TR{alpha} (14). To determine the relative binding affinity of GC-1 for each of the Xenopus TRs, protease protection assays were performed. Protease protection assays have been used previously to both estimate binding affinity and reveal different conformations induced by various natural and synthetic nuclear receptor ligands (24). In addition, the assay is performed at equilibrium and bound and free ligands are not separated, so results tend to accurately reflect the relative transcriptional activation potency of the ligands in question (25). Increasing amounts of T3, the most potent form of TH (Fig. 1, top), induce a major protected band of ~35 kDa in both xTR{alpha} and xTR{beta} (Fig. 2A). The binding curves generated for xTR{alpha} and xTR{beta} were virtually identical (Fig. 2B). Using GC-1, a major protected band of the same size was produced; however, GC-1 bound to xTR{beta} at a concentration at least 10 times lower than that observed for xTR{alpha} (Fig. 2, A and B). Our protease protection assay revealed a preference of GC-1 for TR{beta} over TR{alpha}, which is remarkably consistent with the results of competitive ligand binding assays used for mammalian TRs. Therefore, GC-1 is a TR{beta}-selective ligand for Xenopus TRs.



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  		FIG. 2.
GC-1 binds preferentially to X. laevis thyroid hormone receptor {beta}. A, in vitro translated, 35S-labeled xTR{alpha} or xTR{beta} were incubated with the indicated nanomolar concentrations of T3 or GC-1 and then treated with 4 µg of elastase. The resulting bands were resolved by 12% SDS-PAGE. I, input. B, T3 binding curves for xTR{alpha} (circles) and xTR{beta} (squares) were generated by quantitation of the protected bands shown in panel A and expressed as a percentage of the maximally protected bands. C, GC-1 binding curves for xTR{alpha} (circles) and xTR{beta} (squares) were generated by quantitation of the protected bands shown in panel A and expressed as a percentage of the maximally protected bands. Error bars in panels B and C represent S.D.

 
Activation of X. laevis Thyroid Hormone Receptors by GC-1—We next tested the ability of GC-1 to activate TRs in X. laevis cells using a transient transfection assay. Using X. laevis XLA kidney cells, which express functional TRs, we examined the T3- and GC-1-regulated transactivation properties of the TR{alpha} and TR{beta} ligand-binding domains as Gal4 fusion proteins. The dose-response curves for T3 activation of TR{alpha} and TR{beta} on an upstream activation sequence-based reporter gene were virtually identical, whereas GC-1 activated TR{beta} at lower concentrations than TR{alpha} (Fig. 3, A and B). The leftward shift in the GC-1 dose-response curve was ~10–20-fold at 50% maximal induction, which essentially mirror the protease protection assay results shown in Fig. 2. We next tested the relative ability of GC-1 to activate full-length Xenopus TR{alpha} and TR{beta} by transfecting expression vectors for each subtype individually into the Xenopus XTC fibroblast cell line, which in our hands expresses a low level of functional TRs relative to XLA cells. For example, in the absence of transfected TR, T3 or GC-1 only activated a TH response element-based reporter gene by <2-fold (see insets, Fig. 3, C and D). In response to T3, both TR{alpha} and TR{beta} strongly activated reporter gene transcription. In this case, the maximum level activation is different for TR{alpha} versus TR{beta}, but the EC50 (~10 nM) does not change, as has been reported previously for mammalian TR (26). When GC-1 is used, the maximum activation is similar, but the EC50 value (~20 nM for TR{beta} versus 200 nM for TR{alpha}) is shifted in favor of TR{alpha}. (Fig. 3, C and D). Therefore, in both ligand binding and transactivation assays GC-1 is a TR{beta}-selective ligand for Xenopus as well as mammalian TRs.



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  		FIG. 3.
GC-1 preferentially activates X. laevis TR{beta} versus TR{alpha} relative in transient transfection assays. A and B, an upstream activation stream-containing promoter that drives luciferase reporter gene expression was co-transfected into X. laevis kidney epithelial cells (XLA) along with expression vectors for X. laevis TR{alpha}/Gal4 (circles) or TR{beta}/Gal4 (squares) fusion proteins, and the cells were treated with increasing concentrations of T3 (A) or GC-1 (B). Luciferase activity was normalized to constitutive {beta}-galactosidase activity. C and D, a TH response element-containing minimal promoter driving luciferase reporter gene expression was co-transfected in X. laevis XTC cells with expression vectors for full-length Xenopus TR{alpha} (circles) or TR{beta} (squares), and cells were treated with increasing concentrations of T3 (C) or GC-1 (D). Induction of the TH response element-based reporter gene by T3 or GC-1 without the co-transfection of either TR expression vector is shown in the insets of panels C and D. Luciferase activity was normalized to constitutive {beta}-galactosidase activity. Each data point was determined in triplicate, and error bars represent S.D.

 
Effect of GC-1 on X. laevis Tadpoles—An important advantage of metamorphosis as an assay system is that thyroid hormone can be added directly to the rearing water, causing accelerated effects that mimic what occurs during spontaneous metamorphosis. We treated premetamorphic tadpoles (stages 52 and 53) with increasing doses of both T3 and GC-1. These developmental stages are just prior to the time when the thyroid gland becomes active but after the animals are fully competent to respond to exogenous hormones. We observed the first clear response to GC-1 at 50 nM, where obvious gill resorption and modest tail resorption are induced (Fig. 4A). As the dose of GC-1 is increased, progressively more gill and tail resorption occurs. Very little change is observed in the limbs except for a loss of tissue around the forelimb buds, allowing them to extend away from the body. At high doses of GC-1 (>200 nM) the animals develop a beak-like appearance because of the expansion of Meckel's cartilage. Finally, at 500 nM GC-1 the treated tadpoles are considerably smaller than the controls with an almost complete loss of the tail and gills. Internally, we noted extensive resorption of the intestine beginning above 100 nM GC-1, similar to what is observed with T3 treatments above 10 nM. Like T3, the effects of GC-1 on intact tadpoles are first apparent about 2 days after treatment begins, and most animals survive the 1-week treatments even at the highest doses of GC-1. In some batches of animals we observed limited limb growth and some widening of the brain at concentrations of GC-1 >200 nM, but these effects occurred infrequently and at higher concentration than those required for tail and gill resorption that were always observed.



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  		FIG. 4.
GC-1 strongly induces the loss of larval tissues in premetamorphic (stages 52 and 53) tadpoles, whereas T3 induces both the death of larval tissues and the growth of adult tissues like the hindlimbs and forelimbs. A, top sections, ventral views of tadpoles treated for 1 week with the indicated GC-1 concentrations. G, gills; HL, hindlimbs. Bottom sections, ventral views of tadpoles treated for 1 week with the indicated T3 concentrations. B, dorsal views of the same treated animals as in panel A. Top sections, GC-1-treated animals. Br, brain; G, gills. Bottom sections, T3-treated animals.

 
Our results with GC-1 contrast significantly with the results obtained with the endogenous hormone T3. Low doses of T3 (2 nM) induced hindlimb and forelimb growth and differentiation without significant loss of tail tissue (Fig. 4A). In dorsal views (Fig. 4B), expansion of the brain is readily observed at low T3 doses; we observe a consistent 30–40% expansion of the brain in response to 2 nM T3. Higher T3 concentrations (10 nM) induced limb growth to a greater extent, and gill loss becomes clearly noticeable. Both the growth of adult structures such as the limbs and the resorption of larval tissues such as the tail and gills are apparent at the highest doses of T3 used in these studies (50 nM). A "hunchbacked" appearance is also apparent at these higher T3 doses that is not seen in GC-1 treated animals. This results presumably from the accelerated development of adult dorsal muscle (44). Toxicity was observed when T3 was used in excess of 50 nM, with many animals dying within a few days after treatment. Thus, when GC-1 and T3 dose responses are compared, GC-1 induces death and resorption of larval tissues at lower concentrations than are required to induce limb growth. This is completely opposite to the developmental program induced by the natural hormone T3, where growth responses generally precede larval tissue death and resorption.

The Sensitivity of Tadpole Tails to GC-1 Increases with Increasing TR{beta} Expression—Because higher levels of GC-1 than of T3 were required to cause tissue responses in premetamorphic tadpoles, we next tested whether the efficiency of GC-1 in inducing larval cell death would improve in later developmental stages when TR{beta} levels are significantly elevated. TR{beta} protein expression increases 8–10-fold as tadpoles develop from premetamorphosis (prior to thyroid gland maturation) to metamorphic climax when tail resorption commences in earnest (27). On the other hand, despite a modest increase in TR{alpha} message the TR{alpha} protein levels remain constant throughout metamorphosis. Therefore, we predicted a modest increase in sensitivity to T3 but a large increase in sensitivity to the TR{beta}-selective ligand GC-1 at two developmental stages as follows: (i) premetamorphic stages 52–54 with low TR{beta} levels; and (ii) prometamorphic stages 57 and 58, when TR{beta} levels were nearly maximal but tail resorption had not yet begun. We first established that isolated tail tips from premetamorphic tadpoles would resorb in a T3 dose-responsive and reproducible manner (Fig. 5A). Maximal resorption rates occurred at concentrations above 32 nM T3. We then measured the efficiency of tail resorption in response to increasing T3 and GC-1 at stages 53 and 54 and stages 57 and58. Tails were measured after 6 days of treatment, when the control tails were still healthy and there were clear differences in the response to various doses of hormones (Fig. 5B). Stage 57 and stage 58 tails were modestly more sensitive to T3 (~2-fold at 50% resorption) than were stages 52–54 tails. Strikingly, the stage 57 and stage 58 tails were >12-fold more sensitive to GC-1 than the stages 52–54 tails when the doses required to induce 50% resorption are compared. This increased sensitivity to GC-1 is especially apparent at low doses of GC-1 (50 nM) where there is no response in young tadpoles, but a robust response is observed in older tadpoles.



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  		FIG. 5.
The efficiency of GC-1-induced tail resorption in vitro correlates with TR{beta} but not with TR{alpha} levels. A, tail tips from stages 53 and 54 cultured in vitro show a dose-dependent resorption in response to the indicated concentrations of T3 as compared with untreated tail tips. The extent of tail resorption is expressed as the percentage of tail length remaining in hormone-treated cultures in comparison with cultures treated with vehicle alone. B, tails were obtained from stage 53 and stage 54 tadpoles (open symbols) or stage 57 and stage 58 tadpoles (filled symbols). Squares, T3; circles, GC-1. Error is expressed as S.E.

 
Induction of TH Response Genes by T3 and GC-1—A large collection of TH response genes has been cloned from X. laevis tadpole tails (28) as well as from several other tissues (29–31). Tail TH response genes include rapidly induced early genes that generally encode transcription factors and delayed genes that mostly encode membrane-bound, secreted, or intracellular proteases and extracellular matrix components and receptors (28, 32). Delayed gene expression is more tissue-specific than early gene expression and is especially strong in fibroblasts under the epidermis and between muscle fibers of the tail and in the gills (13). We have analyzed the induction of tail TH response genes by increasing doses of T3 and GC-1, again using stages of development with different levels of TR{beta} expression.

xBTEB (33) is a zinc finger transcription factor, the expression of which is typical of early gene responses to both T3 and GC-1 (Fig. 6, A and B). xBTEB is quite sensitive to T3 because it is induced by as low as 1 nM T3 in stage 54 tadpoles. The induction of xBTEB by T3 at stage 57 is similar. GC-1 induces xBTEB above 100 nM in stage 54 tadpoles; however, at stages 57 and 58 GC-1 is unable to induce xBTEB expression above baseline. Gene 12, TH/bZIP, and TR{beta} itself are other early genes that show a similar dose-response profile to T3 and GC-1 as xBTEB does (data not shown). We next looked at the delayed class of tail response genes, exemplified by the membrane-bound protease fibroblast activation protein {alpha} (FAP{alpha}) (32) (Fig. 6, C and D). First, a 5–10-fold higher level of T3 is required to activate delayed genes like FAP{alpha} than early genes like xBTEB. In response to GC-1, FAP{alpha} mRNA is first induced above base-line at 100 nM with a dose-response curve similar to that of xBTEB. Strikingly, FAP{alpha} becomes more, not less, sensitive to GC-1 in older animals that are expressing more TR{beta}. A clear induction of FAP{alpha} is seen in response to as low as 20 nM GC-1 in stage 57 and stage 58 animals. Other genes in this delayed class that show the same pattern of responses are the secreted matrix metalloproteinase collagenase-3 and the intracellular protease pepE (data not shown). We also noted that the dose-response curves for delayed gene expression closely parallel both the T3 and GC-1 dose-response curves in tail culture assays, further implicating these genes as key players in the process of tail resorption.



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  		FIG. 6.
Northern blot analysis of thyroid hormone response genes induced by T3 and GC-1. Two thyroid hormone response genes, BTEB (A and B) and FAP{alpha} (C and D), were assayed for induction by increasing doses of T3 (A and C, squares) or GC-1 (B and D, circles). RNA was analyzed from the tails of tadpoles at stages 53 and 54 (open symbols) or stages 57 and 58 (closed symbols). Northern data was normalized by stripping the blots and reprobing for constitutive rpL8 expression and is shown as a percentage of the maximum expression for each set of dose-response curves on a given blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A recently developed TR{beta} selective agonist, GC-1, preferentially induces a subset of TH-regulated developmental programs in X. laevis tadpoles, namely the death and resorption of larval tissues. Thus, we provide compelling evidence for different functional roles of the two TR subtypes during metamorphosis, extending earlier expression studies that were strictly correlative (8, 13, 28). Our results agree well with known TR isotype gene expression in that GC-1 induces TR{alpha} rich tissues poorly but is most effective in tissues that have low TR{alpha} levels but that strongly induce TR{beta}. TR{beta} expression in the tail is particularly high in fibroblasts under the skin and around the notochord that also strongly express delayed tail response genes like FAP{alpha}. The pronounced effects of GC-1 on gill and tail resorption are intriguing in light of recent experiments on the expression of TR{alpha} and TR{beta} in various salamander species. Obligatorily neotenic species like Necturus are unable to complete metamorphosis in that the external gills and tail fins of these animals are resistant to TH even though they have fully developed limbs. Interestingly, they express TR{alpha} in those tissues, but there is no detectable baseline or inducible TR{beta} message even though an apparently normal TR{beta} gene exists in the genome (34).

Most tail response genes are induced by similarly high doses of GC-1 in premetamorphic animals (>100 nM) but show very different responses at later developmental stages when TR{beta} is the predominant receptor isotype. Based on these results, we propose that there are two classes of genes in terms of TR subtype control. In young animals, sufficient TR{beta} must be induced via TR{alpha} to activate delayed genes, but other early genes like xBTEB and gene 12 are also being induced in parallel. At later stages only the delayed genes become more sensitive to both T3 and GC-1, indicating that this gene class, at least in the tail, is preferentially controlled by TR{beta}. Our tail culture results are also most consistent with at least a partial requirement for activation for xTR{alpha} to induce sufficient xTR{beta} levels for a response in larval tissues like the tail. Indeed, the xTR{beta} gene contains a strong thyroid hormone response element near its transcription start site (35, 36), and TR{beta} can be induced in tadpoles even in the presence of protein synthesis inhibitors (10). However, there are other possibilities to account for the differences in tissue sensitivity to T3 and GC-1. There may be a less efficient uptake of GC-1 than of T3 from the rearing media in young tadpoles or stage differences in the metabolism of the ligands. In rats, GC-1 accumulates in the liver to a higher degree than in the heart, potentiating its TR subtype-selective properties (16). In this study, we deliberately focused on limb versus tail responses rather than on internal organs because of the equal access of each tissue to the media.

Although our results are consistent with a dominant role for TR{beta} in larval tissue death and resorption, we cannot completely rule out a role for TR{beta} in the formation of adult structures during metamorphosis. For instance, TR{beta} activation may be important in the differentiation of adult cells after several rounds of TR{alpha}-mediated proliferation have occurred. TR{beta} mRNA is detectable in certain remodeling tissues undergoing both resorption and growth such as head cartilage and the intestine (8, 12). In addition, the effects of GC-1 on premetamorphic tadpoles are perhaps more dramatic than expected based on the dose-response profiles of transient transfection assays alone. It is possible that inappropriately timed TR{beta} activation may antagonize TR{alpha} function. The estrogen-inducible progesterone receptor antagonizes estrogen receptor function in the mammalian uterus (37). Another interpretation of our results is that the effects of GC-1 on premetamorphic tadpoles may simply reflect where each isotype is most abundant as the tissue responds. In this model, the two TR isotypes differ quantitatively but not qualitatively. To test this possibility, transgenic approaches are now available in Xenopus, where it should be possible to express xTR{beta} in xTR{alpha}-dominated tissues and vice versa, depending on the availability of appropriate tissue-specific promoters.

There is growing evidence in other animals for isotype-specific functions of nuclear receptors. In insects, ecdysone receptor diversity is generated by alternate promoter usage of the same gene to create different amino-terminal transactivation domains (38). Three ecdysone receptor subtypes in Drosophila melanogaster are expressed in different ratios in different tissues, which correlate with different tissue fates at metamorphosis (38). Specific ecdysone receptor-B1 mutations prevent salivary gland histolysis and development of a subset of imaginal disks that cannot be rescued by overexpression of the ecdysone receptor-A subtype (39). In vertebrates, knockout studies of each TR gene alone and in combination in mice have demonstrated both overlapping and distinct functions for the two TR genes (40). For instance, proper functional development of the inner ear appears to require TR{beta} (41), and TR{alpha} appears to play a more dominant role in TH control of resting heart rate and body temperature (42, 43). However, other processes, such as negative feedback control of the thyroid stimulating hormone gene, were at least partially affected in both TR knockouts (40). It is possible that TR subtype-specific function is more distinct in taxa other than mammals. Certainly, the study of receptor signaling during development is easier in model systems with externally developing embryos. Our findings illustrate the utility of subtype-selective ligands as probes of nuclear receptor function in vivo and demonstrate the potential of natural systems such as amphibian metamorphosis for use as simple bioassays for the development of selective agonists and antagonists of thyroid hormone receptors.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK-52798 (to T. S. S.) and DK-55511 (to J. D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Present address: Dept. of Biochemistry, Dartmouth College, Hanover, NH 03755. Back

|| Present address: St. Louis University School of Medicine, St. Louis, MO 63104. Back

§ To whom correspondence should be addressed: Section of Neurobiology, Physiology, and Behavior, University of California, 1 Shields Ave., Davis, CA 95616-8519. E-mail: jdfurlow{at}ucdavis.edu.

1 The abbreviations used are: TH, thyroid hormone; TR, TH receptor; T3, 3,5,3'-triidothyronine; BTEB, basic transcription element-binding protein; FAP, fibroblast activation protein; x, X. laevis (prefix). Back


   ACKNOWLEDGMENTS
 
We thank Jock Hamilton for photography and the Privalsky laboratory for providing CV-1 cells and advice on their transient transfection. We also thank Phuoc Le, Sherman Lau, and Yvette Chu for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dodd, M. H. I., and Dodd, J. M. (1976) in Physiology of the Amphibia (Lofts, B., ed) pp. 467-599, Academic Press, New York
  2. Tata, J. R. (1998) Cell Res. 8, 259-272[Medline] [Order article via Infotrieve]
  3. Brown, D. D., Wang, Z., Kanamori, A., Eliceiri, B., Furlow, J. D., and Schwartzman, R. (1995) Recent Prog. Horm. Res. 50, 309-315[Medline] [Order article via Infotrieve]
  4. Yaoita, Y., Shi, Y. B., and Brown, D. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7090-7094[Abstract/Free Full Text]
  5. Zhang, J., and Lazar, M. A. (2000) Annu. Rev. Physiol. 62, 439-466[CrossRef][Medline] [Order article via Infotrieve]
  6. Yaoita, Y., and Brown, D. D. (1990) Genes Dev. 4, 1917-1924[Abstract/Free Full Text]
  7. Banker, D. E., Bigler, J., and Eisenman, R. N. (1991) Mol. Cell. Biol. 11, 5079-5089[Abstract/Free Full Text]
  8. Berry, D. L., Rose, C. S., Remo, B. F., and Brown, D. D. (1998) Dev. Biol. 203, 24-35[CrossRef][Medline] [Order article via Infotrieve]
  9. Kawahara, A., Baker, B. S., and Tata, J. R. (1991) Development 112, 933-943[Abstract]
  10. Kanamori, A., and Brown, D. D. (1992) J. Biol. Chem. 267, 739-745[Abstract/Free Full Text]
  11. Fairclough, L., and Tata, J. R. (1997) Dev. Growth Differ. 39, 273-283[CrossRef][Medline] [Order article via Infotrieve]
  12. Shi, Y. B., Su, Y., Li, Q., and Damjanovski, S. (1998) Int. J. Dev. Biol. 42, 107-116[Medline] [Order article via Infotrieve]
  13. Berry, D. L., Schwartzman, R. A., and Brown, D. D. (1998) Dev. Biol. 203, 12-23[CrossRef][Medline] [Order article via Infotrieve]
  14. Chiellini, G., Apriletti, J. W., Yoshihara, H. A., Baxter, J. D., Ribeiro, R. C., and Scanlan, T. S. (1998) Chem. Biol. 5, 299-306[CrossRef][Medline] [Order article via Infotrieve]
  15. Wagner, R. L., Huber, B. R., Shiau, A. K., Kelly, A., Cunha Lima, S. T., Scanlan, T. S., Apriletti, J. W., Baxter, J. D., West, B. L., and Fletterick, R. J. (2001) Mol. Endocrinol. 15, 398-410[Abstract/Free Full Text]
  16. Trost, S. U., Swanson, E., Gloss, B., Wang-Iverson, D. B., Zhang, H., Volodarsky, T., Grover, G. J., Baxter, J. D., Chiellini, G., Scanlan, T. S., and Dillmann, W. H. (2000) Endocrinology 141, 3057-3064[Abstract/Free Full Text]
  17. Lim, W., Nguyen, N. H., Yang, H. Y., Scanlan, T. S., and Furlow, J. D. (2002) J. Biol. Chem. 277, 35664-35670[Abstract/Free Full Text]
  18. Turner, D. L., and Weintraub, H. (1994) Genes Dev. 8, 1434-1447[Abstract/Free Full Text]
  19. Furlow, J. D., and Brown, D. D. (1999) Mol. Endocrinol. 13, 2076-2089[Abstract/Free Full Text]
  20. Nieuwkoop, P. D., and Faber, J. (eds) (1994) Normal Tables of Xenopus laevis (Daudin), Garland Publishing, New York
  21. Tata, J. R., Kawahara, A., and Baker, B. S. (1991) Dev. Biol. 146, 72-80[CrossRef][Medline] [Order article via Infotrieve]
  22. Furlow, J. D., Berry, D. L., Wang, Z., and Brown, D. D. (1997) Dev. Biol. 182, 284-298[CrossRef][Medline] [Order article via Infotrieve]
  23. Shi, Y. B., and Liang, V. C. (1994) Biochim. Biophys. Acta 1217, 227-228[Medline] [Order article via Infotrieve]
  24. Yoh, S. M., and Privalsky, M. L. (2002) Methods Mol. Biol. 202, 129-152[Medline] [Order article via Infotrieve]
  25. Blumberg, B., Bolado, J., Jr., Derguini, F., Craig, A. G., Moreno, T. A., Chakravarti, D., Heyman, R. A., Buck, J., and Evans, R. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4873-4878[Abstract/Free Full Text]
  26. Hollenberg, A. N., Monden, T., and Wondisford, F. E. (1995) J. Biol. Chem. 270, 14274-14280[Abstract/Free Full Text]
  27. Eliceiri, B. P., and Brown, D. D. (1994) J. Biol. Chem. 269, 24459-24465[Abstract/Free Full Text]
  28. Wang, Z., and Brown, D. D. (1993) J. Biol. Chem. 268, 16270-16278[Abstract/Free Full Text]
  29. Shi, Y. B., and Brown, D. D. (1993) J. Biol. Chem. 268, 20312-20317[Abstract/Free Full Text]
  30. Buckbinder, L., and Brown, D. D. (1992) J. Biol. Chem. 267, 25786-25791[Abstract/Free Full Text]
  31. Denver, R. J., Pavgi, S., and Shi, Y. B. (1997) J. Biol. Chem. 272, 8179-8188[Abstract/Free Full Text]
  32. Brown, D. D., Wang, Z., Furlow, J. D., Kanamori, A., Schwartzman, R. A., Remo, B. F., and Pinder, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1924-1929[Abstract/Free Full Text]
  33. Furlow, J. D., and Kanamori, A. (2002) Endocrinology 143, 3295-3305[Abstract/Free Full Text]
  34. Safi, R., Begue, A., Hanni, C., Stehelin, D., Tata, J. R., and Laudet, V. (1997) J. Mol. Evol. 44, 595-604[CrossRef][Medline] [Order article via Infotrieve]
  35. Ranjan, M., Wong, J., and Shi, Y. B. (1994) J. Biol. Chem. 269, 24699-24705[Abstract/Free Full Text]
  36. Machuca, I., Esslemont, G., Fairclough, L., and Tata, J. R. (1995) Mol. Endocrinol. 9, 96-107[Abstract/Free Full Text]
  37. Tong, W., and Pollard, J. W. (1999) Mol. Cell. Biol. 19, 2251-2264[Abstract/Free Full Text]
  38. Talbot, W. S., Swyryd, E. A., and Hogness, D. S. (1993) Cell 73, 1323-1337[CrossRef][Medline] [Order article via Infotrieve]
  39. Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B., and Hogness, D. S. (1997) Cell 91, 777-788[CrossRef][Medline] [Order article via Infotrieve]
  40. Forrest, D., and Vennstrom, B. (2000) Thyroid 10, 41-52[Medline] [Order article via Infotrieve]
  41. Forrest, D., Hanebuth, E., Smeyne, R. J., Everds, N., Stewart, C. L., Wehner, J. M., and Curran, T. (1996) EMBO J. 15, 3006-3015[Medline] [Order article via Infotrieve]
  42. Johansson, C., Gothe, S., Forrest, D., Vennstrom, B., and Thoren, P. (1999) Am. J. Physiol. 276, H2006-H2012[Medline] [Order article via Infotrieve]
  43. Wikstrom, L., Johansson, C., Salto, C., Barlow, C., Campos Barros, A., Baas, F., Forrest, D., Thoren, P., and Vennstrom, B. (1998) EMBO J. 17, 455-461[CrossRef][Medline] [Order article via Infotrieve]
  44. Nishikawa, A., and Hayashi, H. (1994) Dev. Biol. 165, 86-94[CrossRef][Medline] [Order article via Infotrieve]

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