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Molecular Endocrinology, doi:10.1210/me.2002-0274
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Molecular Endocrinology 17 (7): 1395-1409
Copyright © 2003 by The Endocrine Society

Expression Profiles of Novel Thyroid Hormone-Responsive Genes and Proteins in the Tail of Xenopus laevis Tadpoles Undergoing Precocious Metamorphosis

Caren C. Helbing, Kate Werry, Doug Crump, Dominik Domanski, Nik Veldhoen and Carmen M. Bailey

Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6

Address all correspondence and requests for reprints to:Caren C. Helbing, Department of Biochemistry and Microbiology, P.O. Box 3055, Station CSC, University of Victoria, Victoria, British Columbia, Canada V8W 3P6. E-mail: chelbing{at}uvic.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormones (THs) are critical for the growth, development, and homeostasis of many organisms and are necessary for metamorphosis of Xenopus laevis tadpoles. TH-induced metamorphosis requires alterations in the transcriptome and the proteome. However, only a few of the molecular components of this developmental program have been identified and their interrelationship remains unclear. Using a cDNA array comprised of 420 known anuran genes and quantitative PCR, we have identified 93 TH-responsive genes in the tail of premetamorphic tadpoles after exogenous administration of T3. Fifty-three of these mRNA transcripts have not previously been characterized as TH responsive in any species. The gene expression profiles show distinctive temporal patterns with most transcript steady-state levels increasing after induction of metamorphosis. Two-dimensional gel electrophoresis of total protein extracts from the tail shows changes in steady-state levels of many proteins after T3 treatment. Of the up-regulated proteins, 10 were identified by peptide mass mapping. These data identify potential components involved in the regulation of Xenopus tail regression by T3 and begin to address a critical question regarding the interrelationship between the transcriptome and the proteome in TH-dependent developmental processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
METAMORPHOSIS OF THE anuran tadpole is dependent upon the thyroid hormones (THs), T4 and T3. A dramatic increase in endogenous TH from undetectable levels in the premetamorphic tadpole results in the modulation of specific nuclear receptors (TR{alpha} and TRß) and the activation of tissue-specific genetic programs. Subsequent changes in protein expression and activity lead to the complete remodeling of the organism into a frog (1, 2, 3, 4, 5, 6, 7). Virtually every tissue is a target of TH action and the metamorphic response varies from proliferation and differentiation of the limbs to complete apoptosis of the tail. Resorption of the tadpole tail is one of the final events during natural metamorphosis and can be precociously induced by administration of exogenous T3. Modulation of gene expression in response to T3 induction in tail tissue is dependent on the developmental stage of Xenopus laevis tadpoles, suggesting that acquisition of the necessary components for induced regression is a critical step (8). It is possible that differential association of the TR{alpha} and TRß receptors with transcriptional cofactors and accessory proteins may dictate the nature of the genetic program (9, 10). In addition to changes in mRNA expression, protein synthesis also is essential for tail regression (8). However, the relationship between expressed proteins and transcripts during anuran metamorphosis has not been extensively explored.

The expression of a number of genes in X. laevis and Ranid species changes after TH induction (1, 11, 12, 13, 14, 15, 16), and about 35 up-regulated and 10 down-regulated genes have been estimated to be involved in the response to T3 treatment in the tail (8, 17). Using our novel frog cDNA array, we recently identified 79 genes whose steady-state mRNA levels are altered in the Xenopus tadpole tail during natural metamorphosis (18). A similar study defined 26 gene transcripts expressed in the tail of Xenopus tadpoles during precocious metamorphosis that were targets of disruption by the preemergent herbicide, acetochlor (19). In the present study, we use cDNA array and real-time quantitative RT-PCR (QPCR) techniques to identify changes in gene expression in the tadpole tail during T3-induced metamorphosis. In addition, proteomic analysis indicates complexity in the interrelationship between the proteome and transcriptome during tail regression and accentuates the need to examine both components to unravel TH mechanisms of action during development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The presence of constitutively expressed TR{alpha} is critical for the initial response to exogenous TH (20, 21). Subsequent up-regulation in TRß expression appears to contribute to the establishment of tissue-specific genetic programs necessary for metamorphosis (3, 22, 23, 24, 25). To show an appropriate response to T3 induction under our conditions, we used QPCR to determine the mRNA expression levels of TR{alpha} and TRß and the direct response gene encoding the proteolytic enzyme stromelysin-3. The steady-state levels of TR{alpha} mRNA showed no significant change during the exposure time course, whereas the expression of TRß transcript increased approximately 4-fold after T3 treatment (Fig. 1Go). The relative abundance of TR{alpha} mRNA was an order of magnitude greater than TRß mRNA at maximal levels (data not shown), which is consistent with published studies (26). Stromelysin-3 mRNA steady-state levels were also up-regulated approximately 17.5-fold at 48 h (Fig. 1Go). The relative quantities of these transcripts and the timing of induction was similar to previously reported observations (11, 17).



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  		Figure 1. Relative Expression Levels of TR{alpha}, TRß, and Stromelysin-3 mRNA in X. laevis Tadpoles Induced to Undergo Precocious Metamorphosis

Data were obtained by QPCR analyses of mRNA isolated from tails of premetamorphic tadpoles treated with T3 for the indicated times and are presented as fold change relative to the control (0 h). Three independent experiments were performed and error bars represent the SEM. Values displaying statistical significance of P < 0.05 (a) and P < 0.01 (b), and P < 0.001 (c) are indicated.

 
TH-dependent changes in gene expression during resorption of the Xenopus tadpole tail have been identified for a limited number of genes (6, 8, 17). Based on sequence identification, these gene products include transcription factors that may initiate the gene cascade associated with metamorphosis and proteolytic enzymes that function in tissue-specific remodeling events (2, 3, 17). To broaden our identification of genes involved in the TH-dependent response, we employed the frog multispecies analysis of gene expression (MAGEX) cDNA array. This array is comprised of 420 anuran genes whose encoded products display a variety of functions (18, 19). Radiolabeled cDNA prepared from tadpole tail tissue mRNA at each time point was assayed to determine relative gene expression values. A representative region of each array is shown in Fig. 2AGo highlighting some typical gene patterns observed. QPCR on selected gene transcripts was employed to independently confirm the expression patterns observed using the array. Representative gene expression profiles that display the spectrum of responses are shown in Fig. 2BGo.



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  		Figure 2. Multigene Analysis of T3-Induced Metamorphosis Using the MAGEX cDNA Array

A, A representative region of six arrays depicting changes in gene expression profiles in tail tissue from tadpoles treated with T3 for the indicated times. The TH-responsive genes, TH/bZIP and gene 18, are identified by closed and dashed boxes, respectively. B, Representative independent determinations of gene response to TH were obtained by QPCR (bars) and array (line) analyses. Comparison of the fold change response relative to the control for collagenase 4, TH/bZIP, nuclear factor I-B1 (NFI-B1), and meprin, A5-protein, receptor tyrosine phosphatose mu (MAM) domain protein mRNA expression over the indicated times of T3 treatment are shown. Statistical significance of P < 0.05 (a) and P < 0.01 (b) are indicated for the QPCR data.

 
Corrected, normalized densitometric values obtained from MAGEX array analyses were expressed as fold change relative to the control (0 h) and a hierarchical tree cluster was generated. Ninety-one gene transcripts exhibited a change in expression in at least one time point. Eight clusters were identified with correlation coefficients ranging from 0.72–0.90 (Fig. 3Go). Comparison with the literature revealed that 26 genes have been identified as TH responsive in anurans, 8 in mammals, and 2 in both anurans and mammals (Table 1Go) (4, 5, 6, 7, 8, 13, 17, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49). In addition, five gene transcripts (MAPK phosphatase, cdk inhibitor p28, ornithine decarboxylase, NEDD8 homolog and terminal deoxynucleotidyl transferase) did not fall into any of the eight clusters (Fig. 3Go). Their minimum/maximum fold changes relative to the control were 1.0/2.6, 0.4/1.5, 1.0/3.2, 1.0/1.8 and 1.0/2.4, respectively. Of these, ornithine decarboxylase and terminal deoxynucleotidyl transferase are T3 responsive in mammals (27, 28). In total, we have identified 53 genes that have not previously been characterized as TH responsive in any species and have increased the number of known TH-responsive genes common to anurans and mammals by 8.



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  		Figure 3. Cluster Analysis of TH-Associated Gene Expression in Tadpole Tail Tissue of X. laevis Undergoing Precocious Metamorphosis

Fold changes relative to control at each time point were subjected to agglomerative heirarchical clustering analysis. The resultant data were visualized as a heirarchical tree. All gene expression data were normalized to the internal ribosomal L8 mRNA control before clustering. An increase in relative gene expression is depicted in red, whereas a decrease is shown in green. No change is indicated in black. Eight gene clusters exhibiting unique temporal expression patterns are identified by numbered boxes to the right of the diagram. The average expression profile and correlation coefficient of each cluster is also shown. Genes that remain unclustered include: A, MAPK phosphatase; B, cdk inhibitor p28; C, ornithine decarboxylase; D, NEDD8 homolog; and E, terminal deoxynucleotidyl transferase. The GenBank accession nos. for these genes are X83742, U38844, X56316 (Wagner, M. J., and C. C. Helbing, unpublished), and U07803, respectively.

 

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  		Table 1. Gene Identities in Clusters 1–5

 
Cluster 1 is characterized by a single peak of expression at 6 h and includes transcripts encoding three stress proteins and nonmuscle tropomyosin (Fig. 3Go and Table 1Go). Cluster 2 includes transcripts that are down-regulated at all time points after T3 induction with the exception of a transient up-regulation in mRNA levels at 12 h. All genes in this cluster have been characterized as down-regulated by T3 (17, 51). However, the observed transient up-regulation identified by array analysis has not previously been reported. Genes within cluster 3 display a general decrease in expression by 72 h. Of note is the marked transient increase in metallothionein transcript levels observed at 48 h (Fig. 3Go and Table 1Go). Cluster 4 contains gene transcripts whose average expression pattern peaks at 24 h. Cluster 5 includes 13 genes that have an average expression pattern with maximal levels at 24 and 48 h. Predominant transcripts encode proteins important in transcription regulation and signal transduction pathways. Cluster 6 includes 14 genes whose transcripts show distinctive down-regulation at 6 h. Cluster 7 contains seventeen genes that have an average cluster pattern showing marked up-regulation at 48 and 72 h. Of particular note are the known TH-responsive gene 12, TH/basic leucine zipper (TH/bZip), and iodothyronine 5-deiodinase III, which show a very strong induction at 24 h that is maintained up to the 72-h time point (Fig. 3Go and Table 1Go). No change in the level of transcript encoding 5'-deiodinase II was detected (data not shown). The most commonly occurring transcripts in cluster seven encode proteins important in apoptosis and signal transduction. The final gene cluster eight contains 22 genes with an average expression pattern comprising a distinctive peak expression level at 48 h only.

To identify common pathways involved in the tail genetic program, we grouped all genes into functional categories based upon their encoded protein or RNA products and examined their patterns of expression. For the subsequent functional group analysis, we only considered genes that showed signal above background in at least one time point. There appears to be a distinct wave of accumulation of gene transcripts according to functional groups for the up-regulated and down-regulated transcripts. The most commonly represented transcripts whose steady-state levels were changed encode proteins involved in transcription regulation, signal transduction, protein processing, and apoptosis. Because the former two categories had a higher overall representation on the array, we elected to correct for this bias by expressing the frequency of genes up- or down-regulated as a percentage of the total number of genes identified with a signal in each functional group category (Fig. 4Go). Most functional groups display a maximal down-regulation by 6 h and are maximally up-regulated at 48 h (Fig. 4Go). The most striking observation from this analysis is that 30% of the detectable gene transcripts encoding proteins involved in cell structure are down-regulated at 6 h and 26% of the detectable gene transcripts in this category are up-regulated at 48 h (functional group IX; Fig. 4Go). Gene transcripts encoding proteins involved in signal transduction and apoptosis/protein processing (functional groups III and VIII, respectively; Fig. 4Go) show a down-regulation at 6 h (13 and 9%, respectively) followed by an up-regulation by 48 h (16 and 26%, respectively). Up to 17% of gene transcripts that are involved in biosynthesis and metabolism (functional group VI; Fig. 4Go) are elevated between 12 and 48 h, whereas at 6 and 72 h, a similar percentage of transcripts within these groups are down-regulated. Other functional groups that show an up-regulation in more than 15% of gene transcripts encode proteins involved in chromatin structure (functional group II) and transcription regulation (functional group IV; Fig. 4Go).



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  		Figure 4. Expression Profiles of Genes According to Functional Groups

Genes that were identified to be either up- or down-regulated at each time point were grouped according to the function of their encoded gene products. The total number of genes detected during the treatment time course for each functional group is indicated below the figure in brackets. This number served as the denominator in determining the percentage of gene transcripts that were either up-regulated (Up) or down-regulated (Down) in each functional group to eliminate a possible bias inherent in the genes represented on the MAGEX array. Gene transcripts whose functions are unknown were not included (n = 8), as were those encoding proteins involved in the regulation of translation since the levels of these mRNA transcripts did not change throughout T3 treatment (n = 5). Refer to Table 1Go for the definitions of the functional groups.

 
The process of tail regression during metamorphosis requires active protein synthesis (8). To examine the relationship between the transcriptome and the proteome required during this developmental program, we set out to identify some of the proteins whose steady-state levels are altered by T3. Total protein homogenates were isolated from the tails of control and T3-treated tadpoles after 48 h and resolved by 2-D gel electrophoresis. Comparison of Coomassie blue-stained gels revealed 7 protein spots that were reduced and 30 that were increased at 48 h of hormone treatment (Fig. 5Go). Several protein spots that were in sufficient quantity were isolated for peptide mass mapping and their identities are shown in Table 2Go. Of particular note is the identification of two spots corresponding to the zinc finger protein XLCGF3.1, originally identified in Xenopus gastrula-stage embryos, that increase to levels greater than 4-fold in the presence of T3 compared with the control. To examine the relationship between protein and gene transcript levels, we performed QPCR analyses and found that the difference in detected fold changes were within approximately 2-fold for most proteins and their respective transcripts. We did find some notable discrepancies with serum albumin and ß-2 tubulin. Serum albumin gene transcripts were not detected in tail tissue as expected because it is a protein synthesized in the liver and released into the blood (52).



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  		Figure 5. Changes in the Tail Tissue Proteome of X. laevis Tadpoles Undergoing Precocious Metamorphosis

2-D polyacrylamide gels of total protein extracted from tail tissue of tadpoles treated for 48 h with T3 or vehicle control are shown that are representative of two independent experiments. Protein spots that increase in intensity due to T3 treatment are circled on the T3 gel, whereas those that decrease in intensity are circled on the control gel. Proteins used as isoelectric point reference markers are boxed. Spot identities and characteristics are shown in TableGo 2.

 

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  		Table 2. Identification of Proteins and Related Transcripts in the Xenopus Tadpole Tail

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The initiation of the genetic programs governing amphibian metamorphosis is regulated by THs. Exogenous administration of TH to premetamorphic tadpoles induces a precocious metamorphosis and, thus, serves as a useful model for elucidating the mechanisms whereby a single hormonal signal is capable of eliciting multiple cellular outcomes. We investigated the tail-specific response during precocious metamorphosis within NF stages 52–54 because tadpoles at this developmental stage are competent to respond to TH but are functionally athyroid (8). Previous work suggests that the tail resorption program involves a coordinated pattern of TH-dependent gene expression (6, 17, 22, 34). We therefore employed the frog MAGEX cDNA array to identify the expression pattern of 420 transcripts in Xenopus tail tissue in response to T3 treatment. The identified temporal gene expression pattern may be critical for the establishment of normal cellular processes required during resorption of the tail.

Genes found to be modulated by TH fall into two general categories: direct response genes that do not require protein synthesis and delayed response genes whose expression requires the synthesis of factors that mediate TH-dependent action (17). Induction of metamorphosis is dependent upon the TH-responsive nuclear receptors, TR{alpha} and TRß. Transgenic tadpoles expressing a dominant negative form of TR{alpha} showed resistance to a wide range of metamorphic events induced by TH (20). Although TR{alpha} is critical for competence to respond to TH, TRß is thought to establish the genetic programs required for metamorphosis (3, 22, 23). The induction kinetics of TRß identify it as a direct response gene (8, 20, 53). Similar to others, we observed an increase in TRß mRNA levels in the tail within 12 h of T3 treatment. Investigation of the promoter activity of the TRß gene in transgenic X. laevis tadpoles containing a TH response element/green fluorescent protein construct highlights the strong response after exposure of premetamorphic tadpoles to exogenous TH (25). In addition, the presence of TRß protein in tail tissue can be observed approximately 20 h post T3 treatment that is strongly correlated with the up-regulation of TRß mRNA expression (21). QPCR and array analyses indicate that the expression of the majority of T3-responsive genes is altered at 24–48 h following hormone exposure. The promoters of some of these genes have been shown to contain TH response elements (41, 46, 54, 55, 56).

We observed a distinct, transient down-regulation of several gene transcripts within 6 h of T3 treatment (Figs. 3Go and 4Go). Currently, the mechanism of this down-regulation is unclear, but it could reflect a temporary situation of gene repression via an increasing amount of nascent TR protein that is bound in an unliganded state to TH response elements of target genes (3). The levels of T3 at this point may be sufficient to induce TR synthesis but may be insufficient to occupy all available TRs. The inhibitory effect of unliganded TR on gene transcription has been well documented (3, 57). By 48 h, the intracellular hormone levels have increased and thus an increase in ligand-bound TR results in subsequent up-regulation of several target genes. Interestingly, the 48-h time point marks the accomplished transition through the "commitment point" for tail regression. This was defined as a point occurring between 24 and 48 h following T3 treatment, after which withdrawal of the hormone does not halt tail regression in organ culture (8). At this point, protein and RNA synthesis inhibitors also have little effect in inhibiting tail regression (8, 58).

Analysis of the gene expression profiles showed a distinctive pattern that included the tail-specific gene, gene 5 (Ref. 17 and data not shown). Many T3-responsive genes whose expression is restricted to other tissues were not detected in the tail by the MAGEX array. Examples of these include intestinal fatty acid-binding protein (intestine specific) and {alpha}1-microglobulin/bikunin precursor gene (liver specific) (59, 60). Recent work identified TRIP7, a TR-associated protein, to be T3 responsive in the intestine, brain, stomach, body skin, and hindlimb, but not responsive in the tail skin of stage 55/56 tadpoles treated with 5 nM T3 (61). Our results show a transient up-regulation of TRIP7 transcript at 24 and 48 h (Fig. 3Go and Table 1Go). These differences in results could be due to one or a combination of different ages of tadpoles used, T3 exposure dose and examination of tail skin vs. entire tail. We also found that CCAAT/enhancer binding protein-2 mRNA was up-regulated by T3 (Fig. 3Go and Table 1Go) in contrast to observations made in Rana catesbeiana tadpole liver where CCAAT/enhancer binding protein-2 mRNA was refractory to T3 treatment (62). This disparity may be due to differential regulation of this transcript between these tissues.

We identified eight distinct gene clusters within Xenopus tadpole tail based on their differential response after T3 exposure. The majority of clusters display a T3-mediated temporal pattern of gene expression with transient periods of up-regulation during the 72-h observation period. Many of these gene clusters exhibited sequential restricted windows of TH-dependent induction, implying common regulatory mechanisms. Analysis of array data allowed for the identification of 53 genes that were not previously known to be TH responsive. Published data on genes that are TH responsive compare well with our data with the exception of collagenase-4. Previous work has shown that the collagenase-4 gene is up-regulated by T3 in the tadpole tail (31, 37). However, using two independent methods, we show that it is rapidly down-regulated by 6 h (Fig. 2Go). The basis for this apparent discrepancy in results remains unclear, but it may result from the use of different doses of T3.

Hemoglobin switching is a well-known phenomenon that occurs during metamorphosis to allow the organism to adapt from an aquatic to a terrestrial environment. Previous work has shown that during natural and TH-induced metamorphosis larval erythrocytes containing larval globin are replaced by adult erythrocytes expressing an adult globin isoform (18, 40, 63). A decrease in larval ß globin mRNA during natural metamorphosis has previously been reported (18, 36) and we were able to detect this hormone-dependent down-regulation upon T3 treatment (Fig. 3Go and Table 1Go). However, we did not detect an elevation in adult globin mRNA during this time (data not shown). This is not due to an inability to detect adult globin expression because tadpoles undergoing natural metamorphosis show a marked increase in this mRNA (18). These results suggest that adult globin mRNA levels are not T3 responsive, at least during the time frame examined.

We have identified several T3-dependent genes whose expression is affected within 12 h of T3 treatment (Fig. 3Go). Some of these genes have demonstrated prior T3 responsiveness in X. laevis and R. catesbeiana animal models (13, 17, 51). Of note is the up-regulation of the transcripts encoding stress-related proteins such as hsp70, hsp30, ubiquitin, and metallothionein. Both hsp30 and ubiquitin have demonstrated TH responsiveness in tadpole tissues (13, 35). The four genes in cluster two are expressed in the apical cell layer of the tadpole epidermis and are repressed within 16 h of T3 treatment (51). Our results support the observed down-regulated expression for these genes. However, their expression profiles as determined by MAGEX array and QPCR analyses also indicate an initial up-regulation at 12 h following T3 treatment, and therefore, these four genes may not constitute purely repressed genes as such.

Most of the information currently published focuses on the changes in transcript levels during TH-induced metamorphosis with little information concerning the status of the corresponding encoded proteins. The presence of a transcript may not necessarily reflect the protein levels and it does not address critical issues regarding protein functionality. An important case in point is the relationship between TR{alpha} transcripts and protein (21). To further define the transcriptome/proteome relationship during metamorphosis, we have analyzed total tail protein preparations from control and 48 h T3-treated tadpoles using 2-D gel electrophoresis. We were able to identify several T3-responsive proteins that include a number of tubulin isoforms and the ribonucleotide reductase protein R1. The latter binds to {gamma}- and {alpha}/ß tubulin in vitro and promotes microtubule nucleation on the centrosome at the onset of mitosis in Xenopus egg mitotic extracts (64). Other proteins that increased as a result of T3 treatment included desmin, an intermediate filament protein whose mRNA has been found to increase during embryogenesis (65), and creatine kinase, an enzyme important in energy metabolism.

We were also able to identify cytosolic TH binding protein (CTHBP)-pyruvate kinase-muscle isozyme, a protein that is directly involved in thyroid hormone action. In its tetrameric form, this enzyme acts as pyruvate kinase and, as a monomer, it interacts with intracellular TH, thereby modulating intracellular TH concentration. In this manner, CTHBP is thought to be involved in modulating the metamorphic process in different tissues by controlling the level of intracellular thyroid hormones. CTHBP mRNA has been detected in the tail of premetamorphic tadpoles and its level drastically decreases in the resorbing tail (42). We did not see an alteration in the transcript or the protein spot intensity under the conditions used.

Hormone-dependent targeting of gene transcripts that encode enzymes involved in the MAPK signaling cascade (Ras, Raf, MAPKK, MPK1, and MAPK phosphatase; Table 1Go) suggests that posttranslational events are involved in mediating T3-induced apoptosis of the tail. Indeed, previous work in mammalian cells has indicated the importance of MAPK signaling in the TH-induced response (66). The number of proteins identified whose level change in response to T3 treatment is most likely an underestimate because we only looked at the most abundant proteins. It must be emphasized that analysis of subcellular compartments is also necessary to identify additional proteins that may be involved in T3 signaling. From these initial observations, it is evident that more information regarding the status of the proteome including protein steady-state levels and posttranslational modifications needs to be examined in the context of metamorphosis.

In conclusion, we have successfully employed a novel frog MAGEX cDNA array to characterize T3-dependent temporal gene expression in the tadpole tail during precocious metamorphosis of X. laevis and identify 53 gene transcripts that have not previously been identified as TH responsive. The overlap of several genes involved in the T3-induced response of anurans and mammals accentuates the conserved nature of TH signaling between these vertebrates. Indeed, our studies add eight more genes to the common TH-responsive genes. We demonstrate that many of the gene targets have a restricted window of expression after T3 induction that may elucidate the important mechanisms required for tail regression. Comparison of the gene expression and protein profiles in this study with those obtained from tadpole tissues that proliferate in response to T3 will aid in identifying key regulatory points that determine different cellular outcomes to the same hormonal stimulus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
The care and treatment of animals used in this study were in accordance with the guidelines of the Animal Care Committee, University of Victoria. Premetamorphic X. laevis tadpoles (stage 46; Ref.67) were purchased from Xenopus I, Inc. (Dexter, MN) and maintained under natural lighting conditions in a 360L all-glass flow-through aquarium containing charcoal-filtered municipal water at 22 ± 1 C. Tadpoles were fed Nutrafin flakes (Rolf C. Hagen Inc., Montréal, Québec, Canada) daily. For the thyroid hormone-induction analysis, 10–16 animals (NF stages 52–54) were collected from the stock population for each of the treatment time periods (0, 6, 12, 24, 48, and 72 h).

Thyroid Hormone Exposure
Before T3 (Sigma, St. Louis MO) exposure, test animals were acclimatized to laboratory conditions at 22 ± 1 C for 48 h. During the acclimatization and T3 exposure periods, animals were not fed. Ten to 16 NF stage 52–54 (67) tadpoles were exposed to T3 dissolved in dimethyl sulfoxide (Sigma) or a dimethyl sulfoxide solvent control by immersion for 6, 12, 24, 48, and 72 h in 1 liter of pre-aerated water maintained at 22 ± 1 C in glass dishes. The times chosen were intended to include the identified commitment point of the T3-induced response (between 24 and 48 h), after which protein synthesis inhibitors have little effect on induced tail regression (8). T3 was administered at a nominal concentration of 100 nM and the ratio of chemical applicant to water was 1:10,000 (vol/vol). Chemical applications were not renewed during the test period. After the exposures, tadpoles were euthanized in 0.1% tricaine methanesulfonate (MS-222; Syndel Laboratories Ltd., Vancouver, British Columbia, Canada) and tail tissue was collected by removal at the fifth myomere and immediately stored in RNAlater (Ambion, Inc., Austin, TX) at 4 C.

Preparation of RNA
Total RNA was obtained from preserved tadpole tissue using TRIzol reagent as described by the manufacturer (Invitrogen Canada Inc., Burlington, Ontario, Canada). Isolated RNA was subsequently resuspended in ribonuclease-free water and stored at -70 C. Poly(A)+ RNA was isolated from total RNA using the Oligotex mRNA isolation mini kit (QIAGEN Inc., Mississauga, Ontario, Canada).

MAGEX Array Design and Probing
The frog MAGEX cDNA array design is described in detail elsewhere (19). The array contains 420 (exonic or expressed) gene sequences from Xenopus and Rana species. X. laevis ribosomal L8 (GenBank accession no. U00920) cDNA was selected for normalization, as its mRNA transcript levels remain relatively constant under the experimental conditions used (68). Additional housekeeping genes were also included as well as intronic sequences to assess possible genomic DNA contamination. Tadpole tail poly(A)+ RNA (300–400 ng) was used for preparing radiolabeled target cDNA for hybridization as described previously (19). After hybridization, the membranes were exposed to phosphor screens (Molecular Dynamics, Inc., Sunnyvale, CA) for 1 wk. Hybridization signals were collected using a Storm 820 optical scanner phosphor-imaging system (Molecular Dynamics, Inc.) at 50 µm resolution. The resulting image data were converted to a standard 8-bit TIFF file using Photoshop version 5.0 (Adobe Systems Inc., San Jose, CA). Both non-auto- and auto-level images were prepared for analysis to account for signal saturation.

Array Data Analysis
Array image analysis was performed using ScanAlyze version 2.44 (69). Gene expression determinations were done in at least duplicates. Signal intensities were determined from the mean pixel values for each gene and blank spot position and were corrected for noise by subtracting the local median background. Signal intensities that were derived from areas of nonspecific hybridization on the array were not included in the final analysis. A no-signal background value was determined from the average intensity value + 1 SD of blank spot positions across the auto-level dataset. Gene spot positions exhibiting values below the no-signal value were adjusted to this value. The data were then normalized to the mean internal ribosomal protein L8 gene signal. The auto-leveled data were used for further analysis unless signal saturation occurred for a given gene. In this case, the auto-leveled dataset was replaced by the corresponding non-auto-leveled values for all time points. In preparation for agglomerative heirarchical clustering, the background, no-signal level was adjusted to the highest value among all of the blots to prevent false positive changes due to differences in background values. As a data integrity check, ratios were determined for all duplicate positions in both non-auto- and auto-level array images. Gene signals with duplicate ratios greater than 1.67 or less than 0.6 were not considered for final analysis. Only those genes that satisfied all selection criteria at all time points were chosen for further analysis (see the supplemental data published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Otherwise the entire gene set was not used for analysis. Average intensity values were calculated and used to determine the fold changes in expression for each gene relative to control tadpoles. The fold ratios were imported into Cluster (69) and log-transformed before being filtered to values greater than or equal to 0.8 in at least one observation (corresponds to 1.75-fold change). The centered data were then subjected to average linkage clustering to produce a heirarchical cluster tree in Treeview (69).

QPCR
The expression of individual gene targets was analyzed using a MX4000 QPCR system (Stratagene, La Jolla, CA) as described previously (19). Triplicate data obtained for each target cDNA amplification were averaged and normalized to the invariant ribosomal L8 control. Copy numbers were calculated using standard plots that were generated for each target sequence using known amounts of plasmid containing the amplicon of interest. SEM values were calculated using InStat version 3.01 (GraphPad Software, Inc., San Diego, CA).

Total Protein Extraction
Tadpole tails were obtained as described above and each gram of tail tissue was homogenized by two 10-sec pulses at 9500 rpm in a Heidolph DIAX 600 homogenizer (Heidolph Elektro GmbH & Co. KG, Kelheim, Germany) on ice in 3 ml of homogenization buffer. The homogenization buffer consisted of 25 mM HEPES (pH 7.0); 10 mM EDTA, 10 mM ß glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM dithiothreitol (DTT), 100 µM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 1 µg/ml leupeptin, 2 µg/ml antipain, and 300 µg/ml benzamidine. All reagents were from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). The homogenate was clarified by centrifugation at 12,000 x g for 10 min at 4 C, and the protein concentration was determined using the protein assay from Bio-Rad Laboratories, Inc. (Hercules, CA) according to the manufacturer’s instructions. The protein samples were aliquoted and stored at -70 C.

Two-Dimensional (2-D) Polyacrylamide Gel Electrophoresis
The first dimension for protein separation was isoelectric focusing (IEF) tube gels (2.5 mm x 12 cm) that consisted of 9.5 M urea (SigmaUltra urea; Sigma-Aldrich Canada Ltd.), 4% (wt/vol) total acrylamide (3.78% acrylamide; EM Science, Darmstadt, Germany, 0.22% bis-acrylamide; Sigma-Aldrich Canada Ltd.), 4.2% (vol/vol) Pharmalyte 3–10 ampholytes (Amersham Pharmacia Biotech AB, Uppsala, Sweden), 2% (vol/vol) Pharmalyte 5–8 ampholytes (Amersham Pharmacia Biotech AB), 2% (vol/vol) Igepal CA-630 (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada), 0.05% (wt/vol) ammonium persulphate (ACP Chemicals Inc., Montréal, Canada), and 0.07% (vol/vol) N,N,N',N'-tetramethylethylenediamine (TEMED; Sigma-Aldrich Canada Ltd.). The IEF gels were prefocused at 200 V for 15 min, 300 V for 30 min, and 400 V for 30 min. In the IEF running apparatus, the catholyte was 50 mM NaOH (EM Science), and the anolyte was 0.8% (vol/vol) phosphoric acid (ACP Chemicals Inc.). The protein samples were adjusted to 9.5 M urea, 2% (vol/vol) Igepal CA-630, 2% (vol/vol) Pharmalyte 3–10, 2% (wt/vol) DTT, and incubated at room temperature for 2 h. Three hundred micrograms of protein were loaded onto each gel in a 100 µl volume. Samples were electrophoresed for 16 h at 350 V (5600 V·h), then hyperfocused at 800 V for 1 h. The IEF gels were then rinsed in double distilled H2O for 30 sec and equilibrated twice for 15 min in 5 ml of 125 mM Tris-HCl (EM Science) (pH 6.8), 2.5% sodium dodecyl sulfate (Fisher Scientific, Fair Lawn, NJ), 5 mM DTT, 10% (vol/vol) glycerol (EM Science), 0.05% (wt/vol) bromophenol blue (Sigma-Aldrich Canada Ltd.). The second dimension was SDS-PAGE (15 cm x 14 cm x 1.5 mm) composed of a 12% separating gel and a 5% stacking gel. The IEF gels were overlayed with 0.5% (wt/vol) agarose (EM Science) in 125 mM Tris-HCl (pH 6.8), with 0.05% (wt/vol) bromophenol blue, and 2% (wt/vol) sodium dodecyl sulfate. Full Range Rainbow RPN 800 (Amersham Pharmacia Biotech AB) molecular weight markers were used. The gels were electrophoresed at 100 V for 1 h at constant voltage and then at 30 mA per gel at constant current for 4 h. They were then fixed in 50% (vol/vol) ethanol (EM Science), 3% (vol/vol) phosphoric acid at room temperature with shaking overnight, rinsed three times for 30 min in dH2O, equilibrated in 16% (wt/vol) ammonium sulfate (EM Science), 25% (vol/vol) methanol (EM Science), 5% (vol/vol) phosphoric acid for 1 h, and subsequently stained by adding Coomassie brilliant blue G250 (Bio-Rad Laboratories, Inc.) to 0.01% (wt/vol) and shaking at room temperature for 3 d. Gel images were obtained using a DVC digital camera and analyzed using Northern Eclipse software (Empix Imaging Inc., Mississauga, Canada).

Mass Spectrometry Analysis
Spots of interest were excised and the proteins within were reduced, alkylated, and digested with trypsin according to an in-gel digestion protocol (70) with a few modifications. The following sequence was used: the gel pieces were destained in 50% (vol/vol) methanol/5% (vol/vol) acetic acid (ACP Chemicals Inc.), dehydrated with acetonitrile (EM Science), dried, reduced with 50 mM DTT in 100 mM ammonium bicarbonate (Sigma-Aldrich Canada Ltd.) at 56 C for 30 min, alkylated with 100 mM iodoacetamide (Sigma-Aldrich Canada Ltd.) in 100 mM ammonium bicarbonate at 45 C for 30 min in the dark, dehydrated with acetonitrile, hydrated with 100 mM ammonium bicarbonate, dehydrated with acetonitrile, dried, and digested with 20 ng/µl of Sequencing Grade Modified Trypsin (Promega Corp., Madison, WI) in 50 mM ammonium bicarbonate at 37 C overnight. The resulting peptides were extracted out of the gel pieces by incubation in 100 mM sodium carbonate (EM Science), pH 10, for 1 h at 37 C. The peptides were desalted using ZipTip pipette tips containing C18 reversed-phase media (Millipore Corp., Bedford, MA) by washing with 0.1% (vol/vol) formic acid (ACP Chemicals Inc.) and eluting with 50% (vol/vol) acetonitrile/0.1% (vol/vol) formic acid. The eluted peptide sample was applied to the target plate with an equal volume of matrix solution (1% (wt/vol) {alpha}-cyano-4-hydroxycynnamic acid (Sigma-Aldrich Canada Ltd.) in 50% (vol/vol) acetonitrile, 0.3% (vol/vol) formic acid) and allowed to dry. Adrenocorticotropic hormone fragment 1–17 (FW 2093.4), bradykinin fragment 2–9 (FW 904.0) and angiotensin 1 (FW 1296.5) (Sigma-Aldrich Canada Ltd.) in 30% acetonitrile/0.01% formic acid were mixed with an equal volume of matrix solution and placed next to every sample spot on the target plate as external calibrants. Spectra were obtained using a Voyager-DE STR Biospectrometry Workstation matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometer (PE Applied Biosystems, Foster City, CA) operating in positive reflector mode with delayed extraction. Data were manipulated using the Voyager Version 5.1 software with Data Explorer (PE Applied Biosystems). The sample spectra were further internally calibrated using autolytic trypsin peptide peaks. To identify the protein spots from the 2-D gels, the measured mass of the tryptic peptides were searched against the X. laevis entries from the NCBInr03/26/2002 protein database (nonredundant database compiled from a combination of several publicly available protein databases at the National Center for Biotechnology Information, Washington, DC) using the MS-Fit program (University of California, San Francisco; prospector.ucsf.edu). MS-Fit searches were performed with the following parameters: protein molecular mass range of 1,000–100,000 Da, only X. laevis species allowed, one missed cleavage allowed for trypsin digests, cysteines modified by carbamidomethylation, oxidized methionines, and acrylamide modified cysteines as considered modifications, and peptide mass tolerance of ±50 ppm.


   ACKNOWLEDGMENTS
 
We thank Dr. Colleen Nelson, Jason Wilson, and Kim Weigand of the Jack Bell Research Centre (Vancouver, British Columbia, Canada) for array printing and Dr. Bob Olafson and the UVic-Genome BC Proteomics Centre at the University of Victoria (Victoria, British Columbia, Canada) for their expertise. We also thank Rachel Skirrow and Mary Wagner for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) operating and strategic grants (to C.C.H.). C.C.H. is also a recipient of a NSERC university faculty award.

Abbreviations: bZip, Basic leucine zipper; CTHBP, cytosolic TH binding protein; 2-D, two-dimensional; DTT, dithiothreitol; IEF, isoelectric focusing; MAGEX, multispecies analysis of gene expression; QPCR, real-time quantitative PCR; TH, thyroid hormone; TR, thyroid hormone receptor.

Received for publication August 5, 2002. Accepted for publication April 1, 2003.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   TRα  |  TRβ
Ligands:   Thyroid hormone



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