Premetamorphic Tadpoles Research Articles

Effects of Nonylphenol on Rates of Tail Resorption and Metamorphosis in Rana Catesbeiana Tadpoles

Nonylphenol (NP) is a persistent, lipophilic, and toxic chemical that can be endocrine disrupting (estrogenic) at sublethal concentrations. Since amphibian metamorphosis is a hormone-driven process and a delicate balance of hormone levels is required for successful metamorphosis, exposure of larval amphibians to NP might disrupt metamorphic processes. This study tested whether NP exposure influenced rate of metamorphic progression and tail resorption in bullfrog (Rana catesbeiana) tadpoles. Premetamorphic bullfrog tadpoles were exposed for 7 d to one of 3 nominal concentrations of NP (234 μg/L, 468 μg/L, or 936 μg/L) with or without the addition of exogenous 3,3′,5-triiodothyronine (T3). In the absence of exogenous T3, NP significantly increased the rate of tail growth (as measured by tail length) at 936 μg/L. There was no significant effect of NP alone on tail width, limb development, or the process of cranial transformation. When T3 was added to the treatments, increasing NP concentrations were associated with a significant decrease in the rate of cranial transformation, and at the highest dose, the rate of tail resorption was significantly lower than in the controls. Overall, NP had an inhibitory effect on the rate of bullfrog tadpole metamorphic progression and tail resorption. The authors thank the Georgia Basin Ecosystem Initiative (GBEI), Toxic Substance Research Initiative (Health Canada and Environment Canada), Pacific Environmental Science Centre, and Dr. Ken Hall.

Roles of stress hormones in food intake regulation in anuran amphibians throughout the life cycle

Towards understanding the ontogeny of energy balance regulation in vertebrates we analyzed the responses of corticotropin-releasing factor (CRF) and corticosterone to food deprivation in the Western spadefoot toad ( Spea hammondii) at three developmental stages: premetamorphic tadpole, prometamorphic tadpole, and juvenile. Corticosterone responses to 5 days of food deprivation varied among developmental stages. Both pre- and prometamorphic tadpoles increased whole-body corticosterone content with food deprivation, but the magnitude of the response of premetamorphic tadpoles was significantly greater. By contrast, juvenile toads decreased plasma corticosterone concentration. Similarly, brain CRF peptide content tended to increase in food-deprived tadpoles but did not change in food-deprived juveniles. Therefore, there is an ontogenetic difference in the way the hypothalamic–pituitary–interrenal (HPI) axis responds to food deprivation in amphibians. In tadpoles, the HPI axis is activated in response to fasting as is seen in birds and mammals, and may be associated with mobilization of stored fuels and increased foraging. Juvenile toads do not respond to food deprivation by activating the HPI axis, but instead pursue a strategy of energy conservation that involves a reduction in plasma corticosterone concentration.

Development of the respiratory response to hypoxia in the isolated brainstem of the bullfrogRana catesbeiana

The aim of this study was to examine the effects of cellular hypoxia, and the contribution of anaerobic metabolism, on respiratory activity in bullfrogs at different stages of development. Respiratory-related neural activity was recorded from cranial nerve rootlets in isolated brainstem preparations from pre-metamorphic (Taylor-Kollros (T-K) stages VIII-XVI) and postmetamorphic tadpoles (T-K stages XXIV-XXV) and adults. Changes in fictive gill/lung activity in brainstems from pre-metamorphic tadpoles and lung activity in postmetamorphic tadpoles and adults were examined during superfusion with control (98% O(2)/2% CO(2)) or hypoxic (98% N(2)/2% CO(2)) artificial cerebrospinal fluid (aCSF). Iodoacetate (IAA; 100 micromol l(-1)) was used in conjunction with hypoxic aCSF to inhibit glycolysis. Gill burst frequency in pre-metamorphic brainstems did not change over a 3 h exposure to hypoxia and fictive lung burst frequency slowed significantly, but only after 3 h hypoxia. Blockade of glycolysis with IAA during hypoxia significantly reduced the time respiratory activity could be maintained in pre-metamorphic, but not in adult, brainstems. In brainstems from post-metamorphic tadpoles and adults, lung burst frequency became significantly more episodic within 5-15 min hypoxic exposure, but respiratory neural activity was subsequently abolished in every preparation. The cessation of fictive breathing was restored to control levels upon reoxygenation. Neither tadpole nor adult brainstems exhibited changes in neural bursts resembling 'gasping' that is observed in mammalian brainstems exposed to severe hypoxia. There was also a significant increase in the frequency of 'non-respiratory' bursts in hypoxic postmetamorphic and adult brainstems, but not in pre-metamorphic brainstems. These results indicate that pre-metamorphic tadpoles are capable of maintaining respiratory activity for 3 h or more during severe hypoxia and rely to a great extent upon anaerobic metabolism to maintain respiratory motor output. Upon metamorphosis, however, hypoxia results in significant changes in respiratory frequency and pattern, including increased lung burst episodes, non-ventilatory bursts and a reversible cessation of respiratory activity. Adults have little or no ability to maintain respiratory activity through glycolysis but, instead, stop respiratory activity until oxygen is available. This 'switch' in the respiratory response to hypoxia coincides morphologically with the loss of gills and obligate air-breathing in the postmetamorphic frog. We hypothesize that the cessation of respiratory activity in post-metamorphic tadpoles and adults is an adaptive, energy-saving response to low oxygen.

Transgenic analysis reveals that thyroid hormone receptor is sufficient to mediate the thyroid hormone signal in frog metamorphosis.

Thyroid hormone (T3) has long been known to be important for vertebrate development and adult organ function. Whereas thyroid hormone receptor (TR) knockout and transgenic studies of mice have implicated TR involvement in mammalian development, the underlying molecular bases for the resulting phenotypes remain to be determined in vivo, especially considering that T3 is known to have both genomic, i.e., through TRs, and nongenomic effects on cells. Amphibian metamorphosis is an excellent model for studying the role of TR in vertebrate development because of its total dependence on T3. Here we investigated the role of TR in metamorphosis by developing a dominant positive mutant thyroid hormone receptor (dpTR). In the frog oocyte transcription system, dpTR bound a T3-responsive promoter and activated the promoter independently of T3. Transgenic expression of dpTR under the control of a heat shock-inducible promoter in premetamorphic tadpoles led to precocious metamorphic transformations. Molecular analyses showed that dpTR induced metamorphosis by specifically binding to known T3 target genes, leading to increased local histone acetylation and gene activation, similar to T3-bound TR during natural metamorphosis. Our experiments indicated that the metamorphic role of T3 is through genomic action of the hormone, at least on the developmental parameters tested. They further provide the first example where TR is shown to mediate directly and sufficiently these developmental effects of T3 in individual organs by regulating target gene expression in these organs.

Regulation of pituitary thyrotropin gene expression during Xenopus metamorphosis: negative feedback is functional throughout metamorphosis.

Several hypotheses have been proposed to explain the increase and sustained expression of pituitary thyrotropin (TSH) in the presence of elevated plasma thyroid hormone (TH) concentrations at metamorphic climax in amphibians. It has been proposed that the negative feedback of TH on TSH is inoperative until metamorphic climax, and that it is established at this time by the upregulation of pituitary deiodinase type II (DII); DII converts thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)). However, earlier investigators, using indirect measures of TSH, reported that TH negative feedback on TSH was functional in premetamorphic tadpoles. In an effort to understand pituitary TSH regulation during amphibian metamorphosis, we analyzed multiple pituitary genes known or hypothesized to be involved in TSH regulation in tadpoles of Xenopus laevis. Tadpole pituitary explant cultures were used to examine direct negative feedback on TSH mRNA expression. Negative feedback is operative in the early prometamorphic tadpole pituitary and both T(3) and T(4) can downregulate TSH mRNA expression throughout metamorphosis. The expression of both DII and TH receptor betaA mRNAs increased during development and peaked at climax; however, these increases coincided with similar increases in deiodinase type III, which inactivates TH. Moreover, corticotropin-releasing factor (CRF) receptors, CRF binding protein and thyrotropin-releasing hormone receptor type 2 mRNA expression also peaked at climax. Our data suggest that the regulation of TSH is more complex than the timing of DII expression, and likely involves a balance between stimulation of TSH synthesis and secretion by neuropeptides (e.g. CRF) of hypothalamic or pituitary origin, increased pituitary sensitivity to neuropeptides through upregulation of their receptors, and intrapituitary TH levels.

Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis.

Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion-extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5-1 Hz in lumbar ventral roots corresponded to bipedal extension-flexion cycles, while the second, faster pattern (2-5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro. These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.

Molecular cloning of bullfrog prolactin receptor cDNA: changes in prolactin receptor mRNA level during metamorphosis

In amphibian larvae, prolactin (PRL) is known to possess growth-promoting and anti-metamorphic activities. For further understanding of the role of PRL in larvae, bullfrog PRL receptor (bfPRLR) cDNA was obtained from the tail fin of premetamorphic tadpoles by use of the reverse transcription-polymerase chain reaction (RT-PCR) coupled with 5 ′- and 3 ′-rapid amplification of cDNA ends (RACE). The predicted bfPRLR was composed of 617 amino acids, contained a single transmembrane domain, and showed 33–57% sequence homologies with known sequences of vertebrate PRLRs. When bfPRLR was transiently expressed, specific binding of 125I-labeled bullfrog PRL (bfPRL) was observed. By Northern blot analysis, a 3-kb transcript was detected in the tail fin. By RT-PCR bfPRLR mRNA expression was detected mainly in the brain, kidney, skin, and tail throughout prometamorphic and middle climactic periods. The results of an RNase protection assay revealed that the bfPRLR mRNA level in the tail fin increased around the onset of climax (stage XX) and was maintained at a relatively high value at least until mid-climax (stage XXII). It also revealed that bfPRLR mRNA level in the kidney of larvae gradually rose as metamorphosis progressed. The results support the view that PRL in larval period acts not only on the larval organs but also on the organs that are necessary for the adult life to maintain or develop their structures and functions.

Ontogeny of corticotropin-releasing factor effects on locomotion and foraging in the Western spadefoot toad ( Spea hammondii)

We investigated the effects of corticotropin-releasing factor (CRF) and corticosterone (CORT) on foraging and locomotion in Western spadefoot toad ( Spea hammondii) tadpoles and juveniles to assess the behavioral functions of these hormones throughout development. We administered intracerebroventricular injections of ovine CRF or CRF receptor antagonist αhelical CRF (9-41) to tadpoles and juveniles, and observed behavior within 1.5 h after injection. In both premetamorphic (Gosner stage 33) and prometamorphic (Gosner stages 35–37) tadpoles, CRF injections increased locomotion and decreased foraging. Injections of αhelical CRF (9-41) reduced locomotion but did not affect foraging in premetamorphic tadpoles, but dramatically increased foraging in prometamorphic tadpoles compared to both placebo and uninjected controls. Similarly, αhelical CRF (9-41) injections stimulated food intake and prey-catching behavior in juveniles. These results suggest that in later-staged amphibians, endogenous CRF secretion modulates feeding by exerting a suppressive effect on appetite. By contrast to the inhibitory effect of CRF, 3-h exposure to CORT (500 nM added to the aquarium water) stimulated foraging in prometamorphic tadpoles. These tadpoles also exhibited a CORT-mediated increase in foraging 6 h after CRF injection, which was associated with elevated whole-body CORT content and blocked by glucocorticoid receptor (GR) antagonist (RU486) injections. Thus, exogenous CRF influences locomotion and foraging in both pre- and prometamorphic tadpoles, but endogenous CRF secretion in relatively unstressed animals does not affect foraging until prometamorphic stages. Furthermore, the opposing actions of CRF and CORT on foraging suggest that they are important regulators of energy balance and food intake in amphibians throughout development.

Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1

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 TRalpha and TRbeta. GC-1, however, binds to and activates TRbeta at least an order of magnitude better than it does TRalpha. 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 TRalpha levels. The effectiveness of GC-1 in inducing tail resorption and tail gene expression correlates with increasing TRbeta levels. These results illustrate the utility of subtype selective ligands as probes of nuclear receptor function in vivo.

Recruitment of N-CoR/SMRT-TBLR1 corepressor complex by unliganded thyroid hormone receptor for gene repression during frog development.

The corepressors N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) interact with unliganded nuclear hormone receptors, including thyroid hormone (T(3)) receptor (TR). Several N-CoR/SMRT complexes containing histone deacetylases have been purified. The best studied among them are N-CoR/SMRT complexes containing TBL1 (transducin beta-like protein 1) or TBLR1 (TBL1-related protein). Despite extensive studies of these complexes, there has been no direct in vivo evidence for the interaction of TBL1 or TBLR1 with TR or the possible involvement of such complexes in gene repression by any nuclear receptors in any animals. Here, we used the frog oocyte system to demonstrate that unliganded TR interacts with TBLR1 and recruits TBLR1 to its chromatinized target promoter in vivo, accompanied by histone deacetylation and gene repression. We further provide evidence to show that the recruitment of TBLR1 or related proteins is important for repression by unliganded TR. To investigate the potential role for TBLR1 complexes during vertebrate development, we made use of T(3)-dependent amphibian metamorphosis as a model. We found that TBLR1, SMRT, and N-CoR are recruited to T(3)-inducible promoters in premetamorphic tadpoles and are released upon T(3) treatment, which induces metamorphosis. More importantly, we demonstrate that the dissociation of N-CoR/SMRT-TBLR1 complexes from endogenous TR target promoters is correlated with the activation of these genes during spontaneous metamorphosis. Taken together, our studies provide in vivo evidence for targeted recruitment of N-CoR/SMRT-TBLR1 complexes by unliganded TR in transcriptional repression during vertebrate development.

Developmental expression and hormonal regulation of glucocorticoid and thyroid hormone receptors during metamorphosis in Xenopus laevis.

Corticosteroids, the primary circulating vertebrate stress hormones, are known to potentiate the actions of thyroid hormone in amphibian metamorphosis. Environmental modulation of the production of stress hormones may be one way that tadpoles respond to variation in their larval habitat, and thus control the timing of metamorphosis. Thyroid hormone and corticosteroids act through structurally similar nuclear receptors, and interactions at the transcriptional level could lead to regulation of common pathways controlling metamorphosis. To better understand the roles of corticosteroids in amphibian metamorphosis we analyzed the developmental and hormone-dependent expression of glucocorticoid receptor (GR) mRNA in the brain (diencephalon), intestine and tail of Xenopus laevis tadpoles. We compared the expression patterns of GR with expression of thyroid hormone receptor beta (TRbeta). In an effort to determine the relationship between nuclear hormone receptor expression and levels of ligand, we also analyzed changes in whole-body content of 3,5,3'-triiodothyronine (T(3)), thyroxine, and corticosterone (CORT). GR transcripts of 8, 4 and 2 kb were detected in all tadpole tissues, but only the 4 and 2 kb transcripts could be detected in embryos. The level of GR mRNA was low during premetamorphosis in the brain but increased significantly during prometamorphosis, remained at a constant level throughout metamorphosis, and increased to its highest level in the juvenile frog. GR mRNA level in the intestine remained relatively constant, but increased in the tail throughout metamorphosis, reaching a maximum at metamorphic climax. The level of GR mRNA was increased by treatment with CORT in the intestine but not in the brain or tail. TRbeta mRNA level increased in the brain, intestine and tail during metamorphosis and was induced by treatment with T(3). Analysis of possible crossregulatory relationships between GRs and TRs showed that GR mRNA was upregulated by exogenous T(3) (50 nM) in the tail but downregulated in the brain of premetamorphic tadpoles. Exogenous CORT (100 nM) upregulated TRbeta mRNA in the intestine. Our findings provide evidence for tissue-specific positive, negative and crossregulation of nuclear hormone receptors during metamorphosis of X. laevis. The synergy of CORT with T(3) on tadpole tail resorption may depend on the accelerated accumulation of GR transcripts in this tissue during metamorphosis, which may be driven by rising plasma thyroid hormone titers.

Corepressor Requirement and Thyroid Hormone Receptor Function During Xenopus Development

The biologic role of hormonal activation of nuclear receptors is well established. Only recently, however, has the biologic significance of repression begun to be appreciated. Amphibian metamorphosis is marked by dramatic thyroid hormone induced changes, including de novo morphogenesis, tissue remodeling, and organ resorption through programmed cell death. These changes involve cascades of gene regulation initiated by 3,5,3'-triiodothyronine (T(3)). T(3) functions by regulating gene expression through thyroid hormone receptor (TR). TRs are DNA-binding transcription factors that belong to the steroid hormone receptor superfamily. In the absence of a ligand, TRs can repress gene expression by recruiting corepressor complexes, whereas liganded TRs recruit coactivator complexes for gene activation. Corepressor and coactivator complexes induce chromatin remodeling to mediate TR regulation of transcription. The mechanisms of TR action permit a dual function for TRs during development. In premetamorphic tadpoles, when TRs are expressed and T(3) levels are barely detectable, unliganded TRs repress transcription through corepressor recruitment. This TR-mediated repression of target genes is critical for proper larval development, allowing tadpole growth and acquisition of metamorphic competence. In contrast, during metamorphosis, endogenous T(3) causes TRs to activate gene expression, leading to tadpole transformation. Several results also support a role for corepressors during metamorphosis. Corepressor targeted functions, however, are still speculative but may again involve TRs. The requirement of active gene repression at different stages during amphibian development establishes an important biologic role for corepressors.

Metamorphic T3-response genes have specific co-regulator requirements.

Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; tri-iodothyronine), apo-TRs associate with co-repressors to repress transcription, whereas in the presence of T3, holo-TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3-dependent amphibian metamorphosis to analyse how TRs interact with particular co-regulators to differentially regulate gene expression during development. Using chromatin immuno-precipitation to study tissue from pre-metamorphic tadpoles, we found that TRs are physically associated with T3-responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3-response genes. Most importantly, we show that individual T3-response genes have distinct co-regulator requirements, the T3-dependent co-repressor-to-coactivator switch being gene-specific for both co-regulator categories.

Heat hardening as a function of developmental stage in larval and juvenile Bufo americanus and Xenopus laevis

1. We studied heat hardening in tadpoles and juveniles of the temperate Bufo americanus and the subtropical Xenopus laevis by comparing the critical thermal maxima (CTMax) of heat-pretreated animals with the CTMax of control (non-pretreated) animals of the same developmental stages. 2. While premetamorphic tadpoles (Gosner stages 26–43) of both species exhibited heat hardening, metamorphosing B. americanus tadpoles (stages 44–45) were unable to heat harden and, in fact, exhibited a decrease in CTMax following pretreatment while metamorphosing X. laevis tadpoles still exhibited heat hardening. 3. This difference may reflect the more stressful nature of the more extensive metamorphosis of Bufo compared to that of Xenopus.

Expression profiles of novel thyroid hormone-responsive genes and proteins in the tail of Xenopus laevis tadpoles undergoing precocious metamorphosis.

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.

Nuclear receptor corepressor recruitment by unliganded thyroid hormone receptor in gene repression during Xenopus laevis development.

Thyroid hormone receptors (TR) act as activators of transcription in the presence of the thyroid hormone (T(3)) and as repressors in its absence. While many in vitro approaches have been used to study the molecular mechanisms of TR action, their physiological relevance has not been addressed. Here we investigate how TR regulates gene expression during vertebrate postembryonic development by using T(3)-dependent amphibian metamorphosis as a model. Earlier studies suggest that TR acts as a repressor during premetamorphosis when T(3) is absent. We hypothesize that corepressor complexes containing the nuclear receptor corepressor (N-CoR) are key factors in this TR-dependent gene repression, which is important for premetamorphic tadpole growth. To test this hypothesis, we isolated Xenopus laevis N-CoR (xN-CoR) and showed that it was present in pre- and metamorphic tadpoles. Using a chromatin immunoprecipitation assay, we demonstrated that xN-CoR was recruited to the promoters of T(3) response genes during premetamorphosis and released upon T(3) treatment, accompanied by a local increase in histone acetylation. Furthermore, overexpression of a dominant-negative N-CoR in tadpole tail muscle led to increased transcription from a T(3)-dependent promoter. Our data indicate that N-CoR is recruited by unliganded TR to repress target gene expression during premetamorphic animal growth, an important process that prepares the tadpole for metamorphosis.

Metamorphosis-dependent transcriptional regulation of xak-c, a novel Xenopus type I keratin gene.

Anuran larvae transform their epidermis to the adult counterpart during metamorphosis. The major event of this process is the proliferation of larval epidermal basal cells and their differentiation into adult ones. The present study isolated novel type I keratin cDNA dubbed xak-c (Xenopus adult keratin-c) that was exclusively expressed in adult epidermal basal cells. The gene started its expression in the larval epidermis at the onset of metamorphosis. Thyroid hormone (TH) induced the precocious expression of the gene in the epidermis of premetamorphic tadpoles. To study the transcriptional regulation of this gene in relation to epidermal metamorphosis, a 2.8 kb 5'-flanking region of xak-c was cloned and its promoter activity was investigated. Gene constructs were made so as to contain the xak-c promoter region and gene of EGFP or luciferase as a reporter gene and were transfected into various types of cells, which revealed that the 5'-flanking region had an epidermal cell-specific transcriptional activity in both anurans and mammals. Larval skin tissues of Xenopus were transfected with the constructs and cultured in the presence and absence of TH, which showed that the promoter region is responsive to TH, although the region did not contain the consensus TH response element-like sequence. In sharp contrast, the promoter region did not respond to TH in the adult skin, clearly indicating that the cloned region contains specific sequences that respond to metamorphosis-dependent transcription factor(s).

Basic transcription element binding protein is a thyroid hormone-regulated transcription factor expressed during metamorphosis in Xenopus laevis.

Basic transcription element binding protein (BTEB) is a member of the Krüppel family of zinc finger transcription factors. It has been shown that BTEB plays a role in promoting neuronal process formation during postembryonic development. In the present study, the biochemical properties, transactivation function, and the developmental and hormone-regulated expression of BTEB in Xenopus laevis (xBTEB) are described. xBTEB binds the GC-rich basic transcription element (BTE) with high affinity and functions as a transcriptional activator on promoters containing multiple or single GC boxes. xBTEB mRNA levels increase in the tadpole brain, intestine and tail during metamorphosis, and are correlated with tissue-specific morphological and biochemical transformations. xBTEB mRNA expression can be induced precociously in premetamorphic tadpole tissues by treatment with thyroid hormone. In situ hybridization histochemistry showed that thyroid hormone upregulates xBTEB mRNA throughout the brain of premetamorphic tadpoles, with the highest expression found in the subventricular zones of the telencephalon, diencephalon, optic tectum, cerebellum and spinal cord. xBTEB protein parallels changes in its mRNA, and it was found that xBTEB is not expressed in mitotic cells in the developing brain, but is expressed just distal to the proliferative zone, supporting the hypothesis that this protein plays a role in neural cell differentiation.

Developmental Changes in Interrenal Responsiveness in Anuran Amphibians

Basal activity of the hypothalamo-pituitary-interrenal (HPI) axis changes over development in larval amphibians, but development of the responsiveness of this axis to an external stressor has not been studied. We compared developmental changes in whole-body corticosterone content of two anuran amphibian species, Rana pipiens (family Ranidae) and Xenopus laevis (family Pipidae). We also examined developmental changes in the responsiveness of the HPI axis by subjecting tadpoles of different developmental stages to a laboratory shaking/confinement stress and to ACTH injection. We measured whole-body corticosterone content as an indicator of the activity of the HPI axis. Whole-body corticosterone content of R. pipiens remained low during premetamorphosis and prometamorphosis but increased dramatically at metamorphic climax and remained elevated in juvenile frogs. By contrast, whole-body corticosterone content of X. laevis was highest during premetamorphosis, declined at the onset of prometamorphosis, increased at metamorphic climax and remained at climax levels in juvenile frogs. Premetamorphic and prometamorphic tadpoles of both species showed strong corticosterone responses to both shaking stress and ACTH injection. The magnitude and pattern of response differed among developmental stages, with premetamorphic tadpoles of both species showing greater responsiveness to stress and ACTH. Our results show that interrenal responsiveness is developed in premetamorphic tadpoles, suggesting that at these stages tadpoles are capable of mounting an increase in stress hormone production in response to changes in the external environment. Our results also highlight the importance of comparative studies in understanding the development of the stress axis.

Function of thyroid hormone receptors during amphibian development.

Thyroid hormone (T3) plays important roles during vertebrate development (). In humans, T3 is detected in the embryonic plasma by 6 mo and rises to high levels around birth (). During this postembryonic period, extensive tissue remodeling and organogenesis take place. T3 deficiency during human development leads to developmental defects, such as mental retardation, short stature, and in the most severe form, cretinism (,). Likewise, T3 is also critical for amphibian development. It is the controlling agent of anuran metamorphosis, a process that transforms a tadpole into a tailless frog (,). Blocking synthesis of endogenous T3 leads to the formation of giant tadpoles that cannot metamorphose, while addition of exogenous T3 to premetamorphic tadpoles causes precocious metamorphosis. Importantly, most, if not all, organs are genetically predetermined to undergo specific changes, and these changes are organ autonomous. Thus, T3 appears to act directly on individual metamorphosing organs. Such properties have made anuran metamorphosis one of the best-studied postembryonic developmental process at morphological, cellular, and biochemical levels and paved the way for current molecular investigations of the underlying mechanisms.