Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/ NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-l/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are express     

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.     

Expression of type II iodothyronine deiodinase marks the time that a tissue responds to thyroid hormone-induced metamorphosis in Xenopus laevis

The thyroid gland synthesizes thyroxine (T4), which passes through the larval tadpole's circulatory system. The enzyme type II iodothyronine deiodinase (D2) converts thyroxine (T4) to the active hormone 3,5,3'-triiodothyronine (T3) in peripheral tissues. An early response to thyroid hormone (TH) in the Xenopus laevis tadpole is the stimulation of cell division in cells that line the brain ventricles, the lumen of the spinal cord, and the limb buds. These cells express constitutively high levels of D2 mRNA. Exogenous T4 induces early DNA synthesis in brain, spinal cord, and limb buds as efficiently as T3. The deiodinase inhibitor iopanoic acid blocks T4- but not T3-induced cell division. At metamorphic climax, both TH-induced cell division and D2 expression decrease in the brain. Then D2 expression appears in late-responding tissues including the anterior pituitary, the intestine, and the tail where cell division is reduced or absent. Therefore, constitutive expression of D2 occurs in the earliest target tissues of TH that will grow and differentiate, while TH-induced expression of D2 takes place in late-responding tissues that will remodel or die. This pattern of constitutive and induced D2 expression contributes to the timing of metamorphic changes in these tissues.     

A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis

A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role of unliganded TR-induced repression in physiological settings. Here, we analyze the role of unliganded TR in Xenopus laevis development. The total dependence of amphibian metamorphosis upon T3 provides us a valuable opportunity for studying TR function in vivo. First, we designed a dominant negative form of TR-binding corepressor N-CoR (dnN-CoR) consisting of its receptor interacting domain. We confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR-induced gene repression in frog oocytes. Next, we overexpressed dnN-CoR in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative RT-PCR revealed significant derepression of T3-response genes in transgenic animals. In addition, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our earlier model that unliganded TR represses T3-response genes in premetamorphic tadpoles to regulate the progress of development.     

Sequential up-regulation of thyroid hormone β receptor,ornithine transcarbamylase,and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.     

Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis

It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti-LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti-LS antiserum were localized only in the larval epidermal cells. Of 53 and 59-60 kDa acidic proteins that were reactive with anti-LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval-specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS-immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell-enriched splenocytes from normal frogs or in natural killer (NK)-cell-enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti-LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval-specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.     

Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus metamorphosis

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.     

Metamorphosis-associated and region-specific expression of calbindin gene in the posterior intestinal epithelium of Xenopus laevis larva

The present study used a molecular approach toward understanding the mechanism of hormone- and region-dependent remodeling of the small intestine during metamorphosis of Xenopus laevis . A protein spot was noticed on a two-dimensional polyacrylamide gel as a protein whose expression was metamorphic stage- and region-dependent. The protein was identified as the Xenopus homolog (Xcalbindin) of chick calbindin D_28k. Xcalbindin expression in the intestine was restricted to absorptive cells in the posterior part, being detectable at stages 49–61, not detectable at stages 62–63, detectable again at stages 64–66, and finally becoming undetectable in the adult. During spontaneous metamorphosis, the level of Xcalbindin mRNA was significantly increased between stages 57 and 58, dramatically reduced at stage 59, and the mRNA was undetectable from stages 60–63, after which it was weakly re-expressed until the end of metamorphosis. Such up- and down-regulation of Xcalbindin mRNA was induced precociously by exogenous thyroid hormone. These results indicated that Xcalbindin is a specific marker of the differentiated absorptive cells of the intestine. Immunohistochemistry with specific antibodies against Xcalbindin demonstrated that precursor cells of adult intestinal epithelial cells expressed Xcalbindin. Considering these results, the origin of adult intestinal epithelial cells was discussed.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-Lthyronine have profound effects on postenbryonic development of most vertebrates.Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action.Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis,cell death,re-structuring,etc.) in free-living embryos and larvas of most anurans.This article will first describe the salient features of metamorphosis and its control by TH and other hormones.Emphasis will be laid on the key role played by TH receptor (TR),in particular the phenomenon of TR gene autoinduction,in initiating the developmental action of TH.Finally,it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae

Tadpoles that spontaneously arrest development and remain as larvae occur occasionally in Xenopus laevis populations. These non-metamorphosing tadpoles continue to grow, and they develop into grossly deformed giant individuals which come as close as any anurans to being truly neotenic. Giant X. laevis tadpoles that fail to metamorphose lack thyroid glands. In this study, the hypothesis that the tissues of these tadpoles nevertheless remain thyroid hormone sensitive was tested, by exposing isolated tadpole tail tips to exogenous thyroid hormone in tissue culture. The tail tips from giant tadpoles significantly shrank in response to the thyroid hormone treatment, showing that their tissue was still capable of metamorphosis. However, the amount of shrinkage was less than that observed in tail tissue from normal tadpoles. It was hypothesized that complete induction of metamorphosis may not be possible in the giant tadpoles due to a disproportionate growth and development of tissues and organs.     

Loss of reactivity to pan-cadherin antibody in epidermal cells as a marker for metamorphic alteration of Xenopus skin

Pan-cadherin antibodies recognize the conserved C-terminal region of the family of cell-cell adhesion molecules, cadherins, and have a broad spectrum of reactivity to the molecules. In the present study, by immunohistochemistry using an anti-pan cadherin monoclonal antibody (mAb), expression dynamics of cadherins in epidermal tissues were analyzed during metamorphosis of Xenopus laevis. At early stages of development, the anti-pan cadherin mAb detected signals at cell-cell boundaries and in the cytoplasm of both trunk and tail epidermal cells. During metamorphosis, the immunoreactivity decreased in the trunk skin tissue but remained in the tail. At the climax stage, immunoreactivity was observed only in the regressing tail epidermis. The signals disappeared completely from the trunk epidermis, which had already transformed into adult-type tissue. This observation was confirmed by western blot analysis. A specific band was detected in the larval skin, but not in the adult lysate, at approximately 135 kDa in molecular size, corresponding to the molecular mass of cadherins. This different immunoreactivity in larvae and adults was observed in the epidermis of the skin, but not in any other tissues examined, that is, brain, kidney and liver. The immunoreactivity seen in larval epidermal cells was drastically downregulated by thyroid hormone treatment in vitro. These changes of immunoreactivity were specific for the C-terminal region of cadherins, suggesting intracellular alteration of the molecules during metamorphosis, and the anti-pan cadherin mAb can be a marker for larval-type epidermal cells that is applicable to analysis of Xenopus metamorphosis.     

Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone-induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone-induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.