The significance of the latent period in thyroid hormone induced tissue regression during amphibian metamorphosis

Tail fin disks removed from tadpoles of Rana pipiens that were immersed in thyroxine or triiodothyronine for 3 days (referred to as donors) were fused to premetamorphic tail fin blocks that had never been exposed to this hormone (referred to as recipients) so that triplets were formed, consisting of one recipient tail block sandwiched between two donor tail fin blocks. Such recipient tail blocks responded with characteristic resorptive activity within 24 or 48 hr, instead of the minimum 72-hr latent period normally intervening in donor blocks, until shrinkage was initiated in response to triiodothyronine (T₃) or tetraiodothyronine (T₄). The presence of T₃ or T₄ hormone was not required continuously throughout the latent period. Hormone could be withdrawn after 30 hr contact in vivo and after 24 hr contact in vitro without interfering with the rate of tissue regression of tadpole tail fins, suggesting that the “latent period” probably does not coincide with the “critical period” during which subtle biochemical changes presumably occur that precede regression of the tadpole tail during metamorphosis. It is suggested that during the latent period active intermediates may be synthesized that are subsequently transferred from donor tail fins to recipients, thus reducing the latent period of the latter.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.     

Sequential up-regulation of thyroid hormone beta 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 TR beta-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 TR beta 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 TR beta, 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 TR beta and OTC mRNAs.     

Active metabolism of thyroid hormone during metamorphosis of amphioxus

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.     

Alteration of glycoproteins during amphibian metamorphosis

In metamorphosing tadpole liver, the quantitative and qualitative changes in glycoproteins were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and lectin-peroxidase method.     

Apoptosis in amphibian organs during metamorphosis

During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis.     

Programmed cell death during amphibian metamorphosis

The death of different types of cells occurs in regressing or remodeling organs to transform from a tadpole to a frog in both temporally and spatially regulated manners during amphibian metamorphosis. This morphological change is drastic and visible with the naked eye. This review summarizes our current understanding of the basic mechanism of the cell death during the metamorphosis. It focuses in particular on the tail resorption and the remodeling of intestine and skin where programmed cell death is executed by thyroid hormone-signaling through the cell-autonomous response (suicide) and the degradation of the extracellular matrix (murder).     

Hemolytic effect of phenylhydrazine during amphibian metamorphosis

The correlation between the spectral changes in hemoglobin and the severity of anemia induced by phenylhydrazine treatment was studied for the differential sensitivity of amphibians to the drug. Froglets were the most sensitive to phenylhydrazine, followed by prometamorphic tadpoles, adult frogs, metamorphic climax tadpoles, and triiodothyronine-treated tadpoles. The different sensitivities to the hemolytic action of the drug in these animals was rationalized in terms of accessibility, uptake, and detoxication of phenylhydrazine, and a different rate of removal of damaged cells. Postmetamorphic responses were noted for the low uptake of phenylhydrazine by erythrocytes and the loss of facilitated diffusion of 3-O-methylglucose by the erythrocytes of the adult frog.     

Skull development during anuran metamorphosis: III. Role of thyroid hormone in chondrogenesis

Metamorphosis of cranial cartilages in anuran amphibians constitutes one of the most dramatic and extensive ontogenetic transformations in vertebrates. We quantitatively examined the role of thyroid hormone (3,3',5-triiodo-L-thyronine; T3) in mediating gross aspects of this morphological repatterning in the skull of the Oriental fire-bellied toad, Bombina orientalis. T3 was administered via plastic (Elvax) micropellets in three treatment dosages (2.5, 0.25, and 0.025 microgram) and one control dosage (0 microgram) to tadpoles of three Gosner developmental stages--28/29, 30/31, and 32/33; tadpoles were recoved up to 8 d (treatment and control dosages) or 14 d (control dosage) later. Response of larval cartilages to exogenous T3 was dosage dependent but not implant-stage dependent; chondrogenic tissues that participate in metamorphic transformation are competent to respond to T3 well before they normally do. Metamorphic effects of T3 were visible within 2 d; in most treatment groups, the normal metamorphic sequence was two-thirds complete after 8 d. While T3 also induced precocious ossification, the normal temporal relation between bone formation and cartilage transformation was dissociated in experimental groups. Morphological integration between cartilage and bone during cranial metamorphosis is at least partly the result of each tissue responding independently to endocrine factors.