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Endocrinology Vol. 138, No. 7 2989-2997
Copyright © 1997 by The Endocrine Society

ARTICLES

The Type 2 and Type 3 Iodothyronine Deiodinases Play Important Roles in Coordinating Development in Rana catesbeiana Tadpoles1

Kathryn B. Becker, Kristen C. Stephens, Jennifer C. Davey, Mark J. Schneider and Valerie Anne Galton

Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001

Address all correspondence and requests for reprints to: Dr. Valerie Anne Galton, Department of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Borwell Building, Lebanon, New Hampshire 03756-0001. E-mail: Valerie A.Galton{at}Dartmouth.EDU


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In developing Rana catesbeiana tadpoles, the timing of the thyroid hormone (TH)-dependent metamorphic responses varies markedly among tissues. Yet at any one time these tissues are exposed to the same plasma concentration of TH, suggesting that TH action is regulated in part at the level of the peripheral tissues. A major factor in TH action is the intracellular level of the active TH, T3. This level is dependent not only on the plasma concentration of TH (mostly T4) but also on the intracellular activities of the type 2 5'-deiodinase (D2) and the type 3 5-deiodinase (D3), which are responsible, respectively, for generating and degrading T3. (D1 is not present in this species.) To determine whether differential expression of D2 and D3 among tissues could be a significant factor in the coordination of metamorphic events, the ontogenic profiles of the two enzyme activities and corresponding messenger RNA levels in most tissues of R. catesbeiana tadpoles have been documented. The profiles of D2 expression in tail, hindlimb, forelimb, intestine, skin, and eye differed markedly at both activity and messenger RNA levels, but it was notable that expression was invariably highest in a given tissue at the time of its major metamorphic change. D2 expression was very low in brain and heart and did not vary during development. D2 was not expressed in liver, kidney, or red blood cells. With the exception of red blood cells, D3 expression was detected in all tissues studied. Furthermore, it was evident that in tissues that expressed both deiodinase genes, the two expression profiles were comparable, indicating a potential for tight control of intracellular T3 levels.

Direct evidence of the importance of the intracellular conversion of T4 to T3 for TH-dependent metamorphic events was obtained in tadpoles in which endogenous TH synthesis was blocked with methimazole, and the activities of D2 and D3 were inhibited by iopanoic acid. This treatment inhibited metamorphosis. The inhibition could be overcome by the concomitant administration of replacement levels of T3, but not T4.

These results strongly support the view that coordinated development in amphibia depends in part on the tissue-specific expression patterns of the D2 and D3 genes, which ensure that the requisite level of intracellular T3 is attained in a given tissue, regardless of the current level of circulating TH, at the appropriate stage of metamorphosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (TH) is essential for normal development in most vertebrate species. This is perhaps most clearly evident in amphibia, in which metamorphosis does not occur in the absence of a functioning thyroid gland (1, 2). In anurans, TH is required very early in development; although it cannot be detected in plasma of Rana catesbeiana by conventional RIA techniques until midprometamorphosis, it is present in the thyroid gland soon after hatching (3). Low, but critically important, levels must circulate before this stage, because tadpoles hypophysectomized in early premetamorphosis fail to progress beyond this stage unless they are given exogenous TH (4).

Although it is clear that TH is the primary factor responsible for the initiation and accomplishment of individual metamorphic events, very little is known about how these various TH-dependent events are coordinated, not only among the various tissues, but also in different cell types within a given tissue. Furthermore, it is notable that TH-dependent development occurs in different tissues and organs at different stages of the life cycle. For example, differentiation of the hindlimbs takes place primarily during prometamorphosis, whereas the tail does not begin to exhibit its characteristic developmental change, resorption, until the beginning of climax. These marked differences in the timing of TH responses occur even though at any one time all tissues are exposed to the same plasma concentration of TH. This suggests that some regulation of TH action occurs at the level of peripheral tissues.

As in mammals, the major circulating TH in amphibia is T4, which is converted in peripheral tissues to T3 (5), the hormone primarily responsible for TH action (6). Thus, an important factor in determining the extent of TH action in a cell is the intracellular concentration of T3. The intracellular T3 concentration is influenced by many factors, including the cellular uptake of TH and the activities of the iodothyronine deiodinases that metabolize T4 and T3. Three types of deiodinase, type 1 (D1), type 2 (D2), and type 3 (D3), have been identified (7, 8). D1 and D2 catalyze primarily 5'-deiodination (5'D) and, thus, are responsible for the generation of T3. D3 catalyzes primarily inner ring or 5-deiodination (5D), a process that results in the degradation of both T4 and T3 to inactive derivatives (7). Previous studies from this laboratory have shown that both 5D (9, 10) and 5'D (10, 11) activities are present in R. catesbeiana tadpoles. In the tissues studied, some 5D activity was detected throughout development (10), whereas 5'D activity was detected primarily during metamorphic climax; activity was minimal or absent during the early phases of development (10, 11). It was also noted that the characteristics of the 5'D activity in these tissues were typical of the mammalian D2 enzyme (7); values for Michaelis-Menten constant (Km) for rT3 and T4 were in the nanomolar range, and the activity was insensitive to inhibition by 6n-propyl-2-thiouracil (10).

The present study was designed to test the hypothesis that the coordination of metamorphosis among R. catesbeiana tissues resides in part in the differential expression of D2 and D3, with the result that the appropriate intracellular T3 levels for a given tissue can be attained regardless of the levels of plasma TH. To this end we have examined the ontogenic profiles of D2 and D3 expression at both the activity and messenger RNA (mRNA) levels in 10 tissues of R. catesbeiana tadpoles. Determination of the mRNA profiles was made possible by our recent cloning of the R. catesbeiana complementary DNAs (cDNAs) for D3 (12) and D2 (13). The results, which provide strong support for the hypothesis, indicate that there are marked differences in the expression profiles of these genes among tissues, and in tissues that express D2, maximum expression occurs during the phase in which the tissue undergoes its major metamorphic change.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
R. catesbeiana tadpoles (stages V–XXIV) were obtained from Charles D. Sullivan Co. (Nashville, TN). According to the criteria of Taylor and Kollros (14), the tadpoles at stages V–XII were in premetamorphis, those at stages XIII–XIX were in prometamorphosis, and those at stages XX–XXIV were in metamorphic climax. Tadpoles were maintained in distilled water and fed tadpole chow (Carolina Biological Supply Co., Burlington, NC) twice a week. Some tadpoles were maintained in water containing, alone or in combination, 1.0 mM methimazole (MMI), 1.75 µM iopanoic acid (IOP), 0.3 nM T4, and 0.3 nM T3. These compounds were purchased from Sigma Chemical Co. (St. Louis, MO).

Tissue preparations
Tissues, including liver, kidney, intestine, tail, leg (hind and fore), skin, eye, heart, brain, and red blood cells (RBCs), were obtained from the tadpoles as previously described (10, 15). For determination of deiodinase activities, the tissues were homogenized in the assay buffers (5D: 0.25 mM sucrose and 20 mM Tris-HCl, pH 7.6; 5'D: 0.25 mM sucrose, 20 mM Tris-HCl, and 1.2 mM EDTA, pH 7.0) using a Tissumizer (Tekmar Co., Cincinnati, OH). Homogenates were diluted approximately 1:4 (wet wt/vol). The homogenates were centrifuged at 500 x g for 15 min, and the supernatants were stored at -20 C for subsequent assay of 5D or 5'D activity. Total RNA was prepared either by the method of Chirgwin et al. (16) as modified by Schneider and Galton (17) or using a commercial RNA isolation reagent (Tri Reagent, Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions.

5'D and 5D assays in tadpole tissue homogenates
5'D and 5D activities were measured according to previously described methods (18, 19). For the 5'D assays, 1.5 nM [125I]rT3 was used as substrate, and 20 mM dithiothreitol (Sigma) was used as cofactor; for the 5D assays, the substrate was 1 nM [125I]T3, and the cofactor was 50 mM dithiothreitol. [125I]Iodothyronines were obtained from DuPont de Nemours (Wilmington, DE) and were purified by chromatography on Sephadex LH-20 resin (Sigma) before use. Protein concentrations of all samples were determined according to the method of Comings and Tack (20).

Analysis of RNA
Levels of D2 transcripts were determined in samples of tadpole tissue total RNA by slot blot analysis, using the D2 R. catesbeiana cDNA, RC5'DII (13), as probe. Slot blots and radioactive probe were prepared as previously described (17). Each slot contained 10 µg total RNA, the blots were hybridized for 16 h at 42 C, and the final wash was carried out at 60 C. Hybridization signals were detected by autoradiography or detected and quantified using the Molecular Dynamics PhosphorImager (Sunnyvale, CA) and the IPLab Gel computer program (Signal Analytics, Vienna, VA). All blots were stripped and reprobed with the cDNA PR28. This cDNA, provided by Dr. Yun-Bo Shi, NICHD (Bethesda, MD), codes for ribosomal protein L8 (GenBank accession no. U00920), and the expression of the corresponding gene is seemingly ubiquitous and not influenced by either development or TH (21).

D3 transcript levels and, in some cases, D2 were determined by a quantitative reverse transcriptase-PCR (RT-PCR) technique using the Access RT/PCR kit (Promega, Madison, WI). This method, a two-enzyme, single reaction tube system, uses avian myeloblastosis virus RT for first strand cDNA synthesis and Tfl DNA polymerase derived from Thermus flavus for second strand cDNA synthesis and subsequent PCR amplification. The use of a single reaction mixture containing buffer appropriate for both enzymes, 0.5 mM MgSO4, a sense primer, and an antisense primer that serves in the RT reaction and as the downstream primer in the PCR reduces the potential for contamination of the samples. For analysis of D2 transcripts, oligonucleotides based on the sequence of RC5'DII (13) were used as primers: 5'TGCTGCCAACATGGGTCTGCTCA-3' (1–23 bp, sense) and 5'-GGCTTTCCTGAA-GAGCTG-3' (288–306 bp, antisense). The product was probed with the nested oligonucleotide 5'-AAATCCAGCCATGGTCAGTGG-3' (143–163 bp). For analysis of D3 transcripts, oligonucleotide primers were based on the sequence of RC5D (12): 5'-TGCACCTGACCCCCCTTCAT-3' (429–448 bp, sense) and 5'-GGCATTGGTGGGTTGGAAT-3' (573–591 bp, antisense). 5'-TACATCGAGGAAGCCCAC-3' (516–533 bp) was used as probe. The two reactions were carried out sequentially in a thermal cycler by means of linked programs according to the following conditions: RT, 48 C for 45 min and 94 C for 2 min (to inactivate the avian myeloblastosis virus); and PCR, 94 C for 30 sec, 58 C for 1 min, 68 C for 2 min with 1-sec extension at each cycle for 40–45 cycles, and 68 C for 7 min. The optimal concentration of total RNA (1–2 µg) was determined for each tissue.

To quantitate the levels of a specific transcript, a series of RT-PCR reactions containing a constant amount of total RNA and an increasing known concentration of a competitive RNA template was carried out for each RNA sample. The competitor RNA templates for D2 and D3 were prepared according to the recombinant PCR procedure described by Diviacco et al. (22) and used as described by Grassi et al. (23). In brief, the competitor templates were made by inserting a 30-bp nonsense sequence into the native cDNA and subcloning the modified cDNA into pBluescript using the PCR-script kit (Stratagene, La Jolla, CA). The elongated cDNAs were then transcribed in vitro using the T7 RNA promoter sequence and the MEGAscript kit (Ambion, Austin, TX). The products of the RT-PCR reaction were separated by gel electrophoresis using Nusieve agarose (FMC BioProducts, Rockland, ME), transferred to a nylon filter (Nytran, Schleicher and Schuell, Keene, NH), and probed with a nested oligonucleotide as previously described (24). Hybridization signals were visualized and quantified by PhosphorImager analysis and scanning densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Densitometric ratios of standard and sample were determined for each reaction and plotted against the known standard concentration for that particular reaction. The amount of D2 or D3 transcript present in a given RNA sample was calculated by linear regression analysis of the data and extrapolation from a y-intercept value equal to 1, at which point the ratio and standard concentrations are equal.

In some RT-PCR experiments, the products were stained with SYBR Green (FMC BioProducts, Rockland, ME), and the signal was detected by scanning with a FluorImager 575 (Molecular Dynamics) and analyzed using ImageQuant.

Some data were subjected to one-way ANOVA, and statistical differences among groups were determined using Duncan’s multiple range test or were analyzed by Student’s unpaired t test (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ontogenic profiles of 5'D activity and D2 mRNA levels in tadpole tissues
The ontogenic profiles of 5'D activity in six tadpole tissues are shown in Fig. 1Go. The profiles differ greatly from tissue to tissue, but it is notable that levels of 5'D activity in a given tissue are invariably highest during the phase of maximum metamorphic change. Thus, in tail and intestine, two tissues in which the major metamorphic event, resorption, occurs during metamorphic climax, 5'D activity was minimal until the onset of climax, when a marked increase was observed. In hindlimb, which becomes large enough to study by stage XII and undergoes its major differentiation during prometamorphosis, 5'D activity was highest during prometamorphosis and declined to undetectable levels by midclimax. In contrast in forelimb, which develops in the body cavity during late prometamorphosis, emerges at the onset of climax, and continues to differentiate during climax, some 5'D activity was detected in the undeveloped limb before its emergence (data not shown), and activity remained high during climax. In skin and eye, tissues that exhibit TH-dependent changes during prometamorphosis and climax and possibly earlier (1, 26), 5'D was detected at all stages studied. Levels of 5'D activity were very low in brain and heart (data not shown) and did not change during development. As previously reported (10, 11), 5'D activity was not present in liver or kidney at any stage of development, nor was it found in RBCs.



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  		Figure 1. 5'D activity in tissues of tadpoles at different stages of metamorphosis. Bars indicate the mean of values obtained in four to six preparations of tissue from different tadpoles. SEs are indicated.

 
As indicated by slot blot analysis of the corresponding RNA samples, the ontogenic profiles of D2 mRNA transcript levels are very similar to those of the 5'D activities (Fig. 2Go). The blot was probed with RC5'DII. Subsequent reprobing with PR28 resulted in comparable hybridization signals in all slots (data not shown) indicating that the results with RC5'DII were not due to variations in the amounts of RNA applied to the blots. The absence of any significant signal in the liver RNA samples at any stage of development is notable.



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  		Figure 2. Slot blot analysis of D2-related mRNA transcript levels in tissues from tadpoles at different stages of metamorphosis. The blots were hybridized with the D2 probe, RC5'DII, for 16 h at 42 C, and the final wash was carried out at 60 C.

 
As previously reported, Northern analysis of D2-related transcripts in tadpole tail RNA indicated the presence of a major RNA species of approximately 1.5 kilobases (kb) and minor species of 1.8, 2.2, and 7.4 kb (13). The hybridization signal obtained in the present slot blot analyses would have included all of these species. As the signal intensity obtained with the D2 probe in a Northern blot was low, even using RNA from tissues in which 5'D activity was at a maximum, it was not possible to ascertain by Northern analysis that the profiles obtained in the slot blot analysis represented primarily those of the major D2 mRNA species. For this purpose, a quantitative RT-PCR assay was employed. As described in Materials and Methods, reaction mixtures contained, in addition to the RNA of interest, known amounts of a D2 competitor template to allow quantitation of the D2 transcripts. Results obtained with tadpole tail, hindlimb, and forelimb are shown in Fig. 3Go. It is evident that for tail and hindlimb, the profiles of the amounts of the specific D2 mRNA transcripts obtained using the RT-PCR assay are very similar to those obtained by slot blot analysis; more transcripts are present in tail during climax and in hindlimb during prometamorphosis. In the case of the forelimb, which does not emerge until stage XX, slot blot analysis was carried out only on tissue obtained during climax. However, using the Tri reagent (see above), sufficient RNA could be obtained from preemerged forelimb tissue during late prometamorphosis for analysis by RT-PCR. D2 transcripts were present during this phase, and the level doubled during climax. Maximum levels of D2 transcripts in tail, hindlimb, and forelimb were, respectively, 1.8 ± 0.28, 0.6 ± 0.05, and 1.1 ± 0.13 fmol/µg total RNA. Even using this highly sensitive technique, D2 transcripts were undetectable in either liver or kidney RNA (data not shown).



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  		Figure 3. Concentrations of D2 mRNA transcripts in tadpole tissues during prometamorphosis and metamorphic climax, determined using a quantitative RT-PCR assay (details in Materials and Methods). Bars represent the mean of at least four values obtained in RNA samples prepared from different tadpoles. SEs are indicated. Stages XVI–XIX vs. stages XXII–XXIV: tail, P < 0.005; hindlimb, P < 0.001.

 
Ontogenic profiles of 5D activity and D3 mRNA levels in tadpoles tissues
The ontogenic profiles of 5D activity for eight tadpole tissues are shown in Fig. 4Go. 5D activity was detected in all the tissues that expressed 5'D activity, and the most notable feature of each of these tissue profiles is its similarity to that of the corresponding 5'D activity. Thus, like 5'D activity, 5D activity is highest in a tissue during its phase of maximum metamorphic change; it is highest in tail, intestine, and forelimb during climax and in hindlimb during prometamorphosis. Activity was also present in liver and kidney, two tissues that do not express 5'D activity. Activity was minimal in brain and heart and was absent in RBCs (data not shown).



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  		Figure 4. 5D activity in tissues of tadpoles at different stages of metamorphosis. Bars indicate the mean of values obtained in four to six preparations of tissue from different tadpoles. The SEs are indicated.

 
As previously noted, the hybridization signal obtained in RNA from tadpole tissues using the D3 cDNA as probe was weak and could not be quantitated accurately unless gene expression had been enhanced by pretreatment of the tadpoles with T3 (12). This was found to be the case also when the blot used to obtain the data shown in Fig. 2Go was stripped and reprobed with the D3 cDNA. In view of this, D3 transcript levels in RNA from three of the tissues shown in Fig. 4Go (tail, hindlimb, and forelimb) were determined by RT-PCR. In all three tissues, 5D transcripts were present at all stages measured (XVI–XXIV; Fig. 5Go), and levels did not change significantly between stages XVI and XXIII. At the end of metamorphosis a slight rise in the levels was detected in both hindlimb and forelimb. Quantitative RT-PCR was carried out on samples of these tissues obtained during climax. D3 transcript levels in tail, hindlimb, and forelimb during metamorphic climax, expressed in femtomoles per µg total RNA, were, respectively, 1.7 ± 0.11, 0.9 ± 0.41, and 1.6 ± 0.22. These levels are comparable to those of the D2 transcripts (Fig. 3Go).



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  		Figure 5. Relative D3 mRNA levels in tissues of tadpoles at different stages of metamorphosis determined by RT-PCR assay (N.B., samples were not available for tail at stages XXI and XXII). The products of the reaction were separated by gel electrophoresis and stained with SYBR Green. The signal was detected by scanning with a FluorImager 575 and analyzed using ImageQuant. Bars represent the means of values obtained in four to six RNA preparations. Hindlimb, stage XVI vs. XXIV, P < 0.005; forelimb, stage XVI vs. XXIV, P < 0.001.

 
The role of D2 in thyroid hormone action in developing tadpoles
To determine the importance of the intracellular conversion of T4 to T3 for metamorphosis, the effects of inhibiting 5'D and 5D activity in vivo on the ability of exogenous T4 and T3 to induce specific metamorphic events was examined. These studies were carried out in tadpoles in which the synthesis of endogenous TH was blocked with MMI. This hypothyroid tadpole model was chosen to avoid raising plasma TH to levels higher than physiological, a situation that inevitably results when euthyroid tadpoles are given exogenous hormone. We have constantly observed that when metamorphosis is induced by TH in pre- and prometamorphic tadpoles, development is uncoordinated, and the tadpoles ultimately die. The most notable feature in this type of experiment is that the tail begins to resorb before the legs are fully developed, and the latter, although they elongate, remain thin, frail, and turn red. Even when using the lowest effective concentration of TH for longer periods of time, abnormalities in morphological development are apparent, and the animals eventually succumb. By using the hypothyroid tadpole model maintained with and without replacement levels of TH, plasma TH levels can be kept in the physiological range. Hypothyroidism was achieved by maintaining tadpoles in water containing 1 mM MMI. In pre- and early prometamorphic tadpoles, this treatment resulted within a week in complete cessation of leg growth. Other than the absence of development, the tadpoles appeared to be unaffected by MMI even after 4 weeks of exposure. Preliminary studies indicated that leg growth could be restored in the MMI-treated tadpoles if 0.3 nM T3 or T4 was included with the MMI in the bath water. Higher concentrations of TH resulted in leg growth exceeding that of euthyroid untreated tadpoles. The latter, when supplemented with 0.3 nM TH, exhibited an accelerated rate of leg growth.

The study was carried out in prometamorphic tadpoles. In the first experiment the animals were in early prometamorphosis (stages XII–XIV). Leg growth was used as the index of TH action, and the initial leg length at the outset was approximately 10 mm. As the animals were pond-collected, the relatively large SEM was unavoidable. Deiodinase activity was inhibited by adding 1.75 µM IOP to the bath water. As determined by assay in vitro, this treatment resulted in complete inhibition of both 5'D and 5D activities, but, other than inhibiting metamorphosis, it had no apparent effect on the well-being of the tadpoles over the 3-week period of the experiment. The results of this experiment are shown in Fig. 6Go. Leg length in untreated tadpoles nearly doubled during the experiment. Leg growth was slowed and then inhibited in the tadpoles treated with MMI; although there was a statistically significant increase in leg length in this group during the treatment period, this increase occurred during the first week, presumably as a result of residual TH in the animal, and no increase occurred during the subsequent 2 weeks. Leg growth in the MMI-treated tadpoles was restored when either T3 or T4 was included in the bath water. In tadpoles treated with both IOP and MMI, leg growth was completely inhibited, and this inhibition was overcome in part by the inclusion of T3 in the water. In contrast, the inhibition was not overcome when T4 was included in the water.



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  		Figure 6. Effects of T3 and T4 on leg development in tadpoles made hypothyroid with MMI (1 mM in bath water) in the presence and absence of the deiodinase inhibitor, IOP (1.75 µM in bath water). Bars represent the means of values obtained in six to eight tadpoles; the SEs are indicated. Final leg length was determined after 21 days of exposure to the drugs and hormones. *, Significant difference between means of initial and final leg length (P < 0.05).

 
A second experiment was carried out using tadpoles that were in midprometamorphosis (stage XVII), and thus, plasma levels of endogenous TH would have been higher than those in the tadpoles used in the first experiment. Furthermore, it was anticipated that some animals would enter climax during the course of the experiment. Thus, in addition to leg length, three other indices of development were recorded: 1) emergence of the forelimbs, which marks the beginning of climax; 2) shrinkage or loss of the tail fin, a clearly visualized event that marks the beginning of tail resorption; and 3) the change from the oval body shape seen through stage XX to the hour-glass shape that becomes evident during early climax. The results are shown in Table 1Go. In the untreated tadpoles some increase in leg length occurred, and several exhibited forelimbs and/or tail fin loss or change in body shape. In the MMI-treated group, leg growth was not retarded, presumably due to the availability of TH already in the circulation or the thyroid. However, this residual TH was clearly insufficient to support metamorphic climax, because, with the exception of one forelimb in one tadpole, none of the indexes of climax was evident in any of the tadpoles. In contrast, all tadpoles treated with MMI plus either T3 or T4 exhibited at least one index of climax. The combination of MMI and IOP resulted in a retardation of leg growth compared with that of the untreated controls, and none of the tadpoles in this group exhibited any of the indexes of climax. This effect was unchanged in the presence of T4. However, in the presence of T3, not only was leg growth restored, but on the basis of the indexes of climax employed, this group was at least as advanced as the untreated tadpoles.


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  		Table 1. Effects of T3 and T4 on development in hypothyroid tadpoles in the presence and absence of a deiodinase inhibitor, iopanoic acid

 
Effect of thyroid status on D2 and D3 activity in R. catesbeiana tadpoles
The effects of thyroid status on D2 and D3 expression were determined in the prometamorphic tadpoles used in the MMI/TH studies described above. Before prometamorphosis, tadpoles were at the limb-bud stage and provided too little limb tissue for assay. The effects of thyroid status on 5'D activity are shown in Fig. 7Go. In both hindlimb and forelimb, hypothyroidism induced by MMI resulted in a significant increase in 5'D activity. This increase was prevented if T3 was also present in the medium. In the MMI plus T4 group, 5'D activity was significantly lower than that in the untreated tadpoles. As expected, 5'D activity was minimal or absent in tail, which does not exhibit this activity until climax is well established (stage XXI). The data shown in Fig. 7Go and Table 1Go are from the same experiment. Corresponding D2 mRNA levels, as determined by RT-PCR, did not show the same changes. In densitometric units, the mean ± SE were as follows: untreated tadpoles, 732 ± 21; MMI, 425 ± 32; MMI plus T3, 337 ± 32; and MMI plus T4, 503 ± 43. The difference between means in the untreated vs. MMI- plus T4-treated group was statistically significant.



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  		Figure 7. Effect of thyroid status on 5'D activity in prometamorphic tadpole tissues. Bars indicate means of values obtained in seven tadpoles; the SEs are indicated. The data presented in this figure and in Table 1Go were obtained in the same animals. *, Mean value is significantly different from that in the corresponding untreated group (P < 0.05).

 
5D activity determined in the same tissue samples was present, but low, and no clear differences among groups could be detected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroid hormone action is dependent on the interaction of the active TH hormone T3 with its receptor in the nucleus of the target cell (27). It thus follows that the extent of TH action in a cell must be influenced by the intracellular concentrations of both T3 and its receptors. We and others have demonstrated the presence of T3 receptors in several tissues of Rana catesbeiana tadpoles, including tail (28, 29), liver (30, 31), and RBCs (32, 33), and have shown that they are present early in development (6, 32) and that receptor number increases in some tissues as the tadpole approaches metamorphic climax (29, 32, 34). This increase in receptor number undoubtably contributes substantially to the regulation of metamorphosis. However, the importance of TH levels is evident from the failure of the tadpoles to metamorphose in the absence of TH and the marked dose dependency of the TH-induced metamorphic responses (1). Furthermore, it is well established that plasma TH levels, primarily T4, are low in premetamorphosis, increase gradually during prometamorphosis, and then increase much more rapidly during climax, reaching a peak at stage XXII (32, 35). What is not clear is how, in this setting, different tissues can metamorphose at different stages of development. We hypothesize that the answer lies in part in the differential tissue expression of the D2 and D3 genes.

The ontogenic profiles of 5'D activity described herein strongly support this hypothesis. This is most evident when the 5'D activity profiles obtained in the tail and the limbs are compared. 5'D activity is highest in hindlimbs during prometamorphosis, the phase in which they undergo differentiation; thereafter, activity declines to very low levels. On the other hand, tail does not exhibit 5'D activity and does not start to resorb before the onset of climax. Forelimb exhibits 5'D activity and undergoes differentiation during both phases. The 5'D activity profiles in other tissues add support for the hypothesis. Intestine, which like tail undergoes its major transformation during climax, including a 75% reduction in length (1), also does not exhibit 5'D activity until climax. In skin and eye, which undergo TH-dependent changes during most of the larval stages (1, 36), 5'D activity is present at low levels at all stages studied. As previously reported, no 5'D activity was detected in liver or kidney at any stage of development (10, 11).

The ontogenic profiles of 5D activity were somewhat unexpected. Previous in vitro studies had revealed that 5D activity in tadpole liver was highest in pre- and prometamorphosis and then declined to very low levels during climax (9). In contrast, 5'D activity in tail and intestine was minimal until climax, when it increased dramatically (11). When these findings were considered in the context of an earlier observation that [125I]T3 could not be detected in tadpole plasma after the injection of [125I]T4 until late prometamorphosis (37), it appeared likely that the major role of the 5D system was to prevent any accumulation of T3 during the early phases of development. The present data do not contradict those in the previous report with respect to the liver, as the D3 profile obtained in liver is similar to that reported previously (9). However, the additional data obtained in other tissues indicate that the role of the 5D system is not limited to the early phases of metamorphosis. Indeed, it is notable that in all tissues in which 5'D activity is present, the 5D and 5'D activity profiles are quite similar, indicating that 5D activity, like 5'D activity, is highest during the phase that these tissues are undergoing their major metamorphic changes. This suggests that the two systems act in a coordinated push-pull fashion to provide a very tight regulation of intracellular T3 levels at the time of maximum metamorphic change, in much the same way as has been suggested for mammalian fetal brain (8). Given the deleterious effects of even relatively low levels of exogenous hormone on metamorphosis (see above), this coordination of the two systems is likely to be a critical feature of metamorphosis.

It is not clear why 5'D activity is absent in liver and kidney; to date, amphibia are the only species in which the absence of 5'D activity (D1 or D2) in hepatic tissue has been reported (7, 38, 39, 40, 41). Both tissues are known to undergo major TH-dependent metamorphic changes. These changes have been widely studied in tadpole liver (1, 42) and to a lesser extent in kidney (1), and they take place during prometamorphosis and climax. In the absence of a 5'D system, these tissues must be dependent on the plasma for T3, even during prometamorphosis when plasma T3 levels are very low (35). Yet, TH-dependent metamorphic changes take place spontaneously in liver at this time (43). The reason for this difference in tissue response is not clear, but one possibility is that liver and kidney contain a transport system to facilitate the uptake of T3. There is ample precedence for this in other species, and the topic has been reviewed (44). Furthermore, there is evidence that the uptake of T3, but not T4, into tadpole RBCs is facilitated by a carrier-mediated system (45). This is of interest because tadpole RBCs, like liver, do not contain 5'D activity, yet TH-dependent metamorphic effects occur spontaneously in RBCs during prometamorphosis (32) and can readily be induced by TH both in vivo (17) and in vitro (46). As suggested for the tissues in which 5'D activity is present, the 5D system is probably present in liver and kidney to prevent excessive accumulation of T3.

The present data offer some insight into how 5'D activity is regulated during development. The ontogenic profiles of D2 mRNA in the tadpoles tissues studied correspond very closely to the 5'D activity profiles, suggesting that changes in activity are secondary to changes in D2 mRNA levels. Although a change in the latter could be due in part to a change in the rate of mRNA degradation, it seems more likely that the rapid change from a minimal to the readily detectable level of D2 mRNA seen in tail and intestine near climax is the result of an increase in transcription of the D2 gene. How the differential tissue expression of this gene during development is achieved remains to be determined.

5'D activity is also influenced in some circumstances during development by thyroid status. In prometamorphic tadpoles, 5'D activity was enhanced in hind- and forelimbs of tadpoles made hypothyroid by treatment with MMI. This increase was not observed in tadpoles maintained in MMI plus T3, and 5'D activity was significantly decreased in tadpoles maintained in MMI plus T4. These changes were not reflected in corresponding changes in D2 mRNA levels, suggesting that the effect occurred primarily at a posttranslational level. These effects of thyroid status on 5'D activity resemble those described for mammalian D2 activity (7), including the finding that T4 was more potent than T3 in inhibiting 5'D activity (47). It is important to differentiate the present studies, in which tadpoles were made hypothyroid and given replacement doses of TH, from previous studies in which euthyroid tadpoles were subjected to TH-induced metamorphosis. In the latter case, treatment with TH (20 nM) results in the tadpoles entering climax after 10–14 days, and as is the case during spontaneous metamorphosis (11), this is accompanied by an increase in 5'D activity in tail, skin, and gut (10). However, acute administration of TH (10 nM for 4 days) has no effect on 5'D activity (12). The present study was designed to examine the effects of the hormones when present at concentrations in the physiological range in tadpoles.

How 5D activity is regulated during spontaneous metamorphosis is not clear. Previous studies have shown that the amphibian D3 gene is up-regulated by TH (12, 48). However, in R. catesbeiana the increase in D3 expression induced by treatment of tadpoles with exogenous TH is transient; it is maximum at 3 days, but by 10 days expression has returned to pretreatment levels (12). How this phenomenon influences D3 expression in the physiological setting where plasma TH levels are rising continuously until late climax is not known. In both the previous (12) and the present study, D3 mRNA levels were too low to quantitate accurately by Northern or slot blot analysis unless tadpoles were treated with TH. Thus, in the present study levels at different stages were compared using RT-PCR. D3 mRNA levels were comparable in tail and limb at all stages studied (XVI–XXIV), except for a small but significant increase in D3 levels in both types of limb when mean values obtained at stages XVI and XXIV were compared. It is possible that this increase results from the dramatic increase in plasma TH levels that occurs in midmetamorphic climax (32, 35). Before stage XVI, D3 mRNA levels were generally extremely low. What is clear from this study is that in hindlimb, levels of 5D activity and D3 mRNA do not correlate. It is also noted that the increases in D3 mRNA level in fore- and hindlimbs at stage XXIV were not reflected by an increase in D3 activity. This suggests that factors other than D3 gene expression per se are influencing D3 activity at least in these tissues.

The ontogenic patterns of expression provide strong, but indirect, support for the hypothesis that the D2 and D3 enzymes play an important role in metamorphosis. To obtain more direct evidence, experiments were carried out to determine whether the physiological action of T4 could be prevented in vivo under conditions in which 5'D activity is inhibited by IOP. IOP is known to be a potent inhibitor of 5'D activity (7), and it has been reported that in hypothyroid rats, the suppressive effect of T4 on plasma TSH levels was not observed if the rats were exposed to IOP; suppression of plasma TSH by T3 was unaffected (49, 50). Similarly, in rat brown adipocytes, a tissue in which receptor occupancy is strongly influenced by local D2 activity (51), the effect of T4, but not that of T3, was abolished in the presence of IOP (52). Both 5'D and 5D in tadpoles have been shown to be inhibited both in vitro (9, 10) and in vivo (5) by IOP. The present experiments were carried out in tadpoles made hypothyroid by treatment with MMI. Again, this feature of the experiments was important because it permitted the use of exogenous T4 and T3 in replacement and, hence, physiological levels, thus avoiding the complication of an unphysiological increase in receptor occupancy and, hence, TH action by T4 per se. We have previously shown that T4 does not have to be converted to T3 to be active in this species (46). In these studies two major observations were made. First, it was evident that the retardation of metamorphosis was greater in tadpoles exposed to both IOP plus MMI than in those exposed to MMI alone. As MMI only inhibits the synthesis of TH, it would not be expected to inhibit the effects in peripheral tissues of any residual, previously synthesized TH. However, when IOP is present with MMI, conversion of T4 to T3 in peripheral tissues would also be inhibited, thus limiting the effects of any residual circulating T4. The finding in both experiments that IOP further reduced TH-dependent development in MMI-treated tadpoles is, therefore, consistent with an important role for 5'D activity. Second, the inhibitory effects of IOP on TH-dependent metamorphic events were reversed by T3, but not by T4. The most direct explanation for this difference in the actions of the two hormones is that T4 is ineffective in the presence of IOP because it cannot undergo the necessary conversion to T3 in the tissues. T3, on the other hand, requires no such activation; thus, its action is not inhibited. Indeed, as IOP inhibits also 5D activity, an enhancement of T3 action might be expected depending on the level of 5D activity in the tissue under study. No enhancement was observed in the first experiment using the younger tadpoles, but in the second one, the effect of T3 on tail fin resorption was clearly greater in the presence of IOP than in tadpoles treated with MMI and T3 alone. Although it has been reported that IOP can compete with TH for its receptor (53, 54), an effect that might also reduce the physiological effects of TH, this does not seem to be a major factor in the present studies. The mean increase in leg length was somewhat lower in tadpoles treated with MMI/IOP/T3 than in those treated only IOP/T3, but it was only marginally so in the second experiment, and in neither case was the difference significant. Furthermore, in the second experiment some of the effects of T3 were actually enhanced by IOP.

In summary, these findings strongly support the contention that in many tadpole tissues, local generation of T3 by the D2 system is essential for the successful accomplishment of metamorphosis, and thus, a key feature of metamorphosis is the differential tissue programming of D2 and possibly also D3 gene expression during the life cycle of this species.


   Footnotes
 
1 This work was supported by USPHS Grants HD-09020 and HD-27706. Back

Received February 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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