Thyroxine-induced Metamorphosis Research Articles

Thyroxine-induced metamorphosis in the axolotl (Ambystoma mexicanum).

The axolotl (Ambystoma mexicanum) has remained an important model for regeneration and developmental biology for over a century. Although axolotls in captive-bred colonies usually exist in an aquatic form, they retain the ability to undergo metamorphosis following exposure to thyroid hormone. Here we present a robust method for inducing metamorphosis in adult axolotls that results in high survivability and produces terrestrial animals that can be maintained in long-term captivity.

Calbindin Immunoreactivity in Purkinje Cells of the Bullfrog Cerebellum during Thyroxine-Induced Metamorphosis

Calbindin-immunoreactive Purkinje cells were identified in the cerebella of frog tadpoles that had been treated with thyroxine to accelerate metamorphosis. The dorsal part of the cerebellar plate contained the full complement of Purkinje cells which were all CaBP-immunoreactive, while in the ventral part of the cerebellum Purkinje cells acquired CaBP-immunoreactivity only after several days of thyroxine treatment. The ventral group of Purkinje cells was separated from the dorsal group by a distinct gap, which is the site of a shallow sulcus in adult frogs. Additionally, following thyroxine treatment, the numbers of CaBP-immunoreactive Purkinje cells in the ventral group were only half the numbers seen in frogs that metamorphosed spontaneously. We suggest that the variation in the CaBP-immunoreactivity of the dorsal and ventral groups of Purkinje cells, along with the gap in the Purkinje cell layer between the two groups, may be indicative of two distinct populations of Purkinje cells, with distinct patterns of generation, maturation, and perhaps, origin and connectivity, in the cerebellum of frogs.

Ontogeny of immunoglobulin expression in the Mexican axolotl.

The ontogeny of immunoglobulin (Ig) synthesis was followed at both cellular and serological levels in the Mexican axolotl (Ambystoma mexicanum) using polyclonal antibodies recognizing all Ig molecules and a set of monoclonal antibodies (Mabs) specific for the C mu and Cv heavy Ig chain isotypes and for the light chain constituents shared by IgM and IgY molecules. Clusters of IgM- and of IgY-synthesizing lymphocytes, often located in separate sites, are first present in spleen sections of 7-week-old 25 mm larvae, about one month after differentiation of the spleen anlage (stage 39-40). In 12-week-old 30-35 mm larvae, the relative proportion of IgM- and IgY-synthesizing cells in the spleen is the same as that in adult animals. However, a marked enhancement of the spleen B cell compartment occurs from 5 to 9 months when Ig-positive cells represent about 88% of the lymphocytes population compared to 60% in adults. No structures equivalent to B cell germinal centers were observed at any stage of the spleen differentiation and cells, although often clustered in small groups, remain dispersed in the entire organ. The relative proportions of IgM and IgY B cells throughout the spleen remain constant during development (about 1 IgY+ cell for 5-6 IgM+ cells) and IgM molecules are first detected in the serum of 2.5-month-old larvae. The enhancement of the serum IgM level correlates well with the absolute number of IgM+ cells in the growing spleen. IgY molecules cannot be detected in the serum before the 7th month but their level quickly increases to reach about 60% of the adult value at 10 months. Thyroxine-induced metamorphosis or hyperimmunization of 4- to 6-month-old larvae had no effect upon the temporal expression of the Ig classes in serum.

Connections between the nucleus isthmi and the tectum in larval and post-metamorphic axolotls.

The nucleus isthmi (NI) is the primary relay for the frog's ipsilateral visuotectal projection. Using electrophysiological methods, ipsilateral visuotectal activity has been recorded in thyroxine-treated, postmetamorphic axolotls but not in larval axolotls. In order to determine whether changes in isthmotectal projections are responsible for this change in electrophysiological responsiveness, we have investigated the connections between the tectum and the NI using horseradish peroxidase. Our results indicate that the axolotl's isthmotectal pathways are strikingly similar to those of the frog NI, and that the NI sends bilateral projections to the tecta in both larval and thyroxine-treated, postmetamorphic axolotls. Thus, the anatomical connections underlying the ipsilateral visuotectal projection are present during larval stages, despite the lack of electrophysiological evidence for the larval ipsilateral visuotectal projection. We hypothesize that thyroxine-induced metamorphosis produces changes in the terminal arborizations of the crossed isthmotectal projection which allow them to be detected by presynaptic electrophysiological techniques.

Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver

Thyroxine-induced metamorphosis of the tadpole to the frog ( Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes—carbamyl-phosphate synthetase I and argininosuccinate synthetase—were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phospho enolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phospho enolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin.

Granule cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Granule cell maturation in the cerebellum of bullfrog tadpoles was studied during both spontaneous and thyroxine-induced metamorphosis by using electron microscopy and Golgi-impregnated preparations. The production of cerebellar microneurons, a majority of which are granule cell precursors, was quantitatively compared during spontaneous and thyroxine-induced metamorphosis by using stereological methods and biochemical measurements of DNA. Granule cell migration and differentiation appeared morphologically similar during spontaneous and thyroxine-induced metamorphosis. In both instances, granule cells migrated tangentially along the pial surface, migrated into the internal granular layer, developed dendritic arbors, and formed synaptic contacts with the processes of Golgi cells and with mossy fibers. These events are similar to developmental processes that have been described in detail in other animals. Quantitative stereological measurements demonstrated similar overall patterns of change during spontaneous and thyroxine-induced metamorphosis. Most notably, increases in the volume of the external granule layer correlated with increases in the relative and total amounts of DNA. However, measurements of total DNA were consistently reduced during the period of accelerated change that occurs in thyroxine-induced metamorphosis, although external granular layer volume was greater in these tadpoles after 2 and 3 weeks of thyroxine treatment than in spontaneously metamorphosing tadpoles. While granule cell development in the frog is largely dependent on thyroid hormone, differences between thyroid-hormone-induced and spontaneously metamorphosing tadpoles suggest that normal patterns of cerebellar development are also dependent on events that occur in premetamorphic tadpoles in the absence of thyroid hormone.

Stellate cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Stellate cell development was studied in the bullfrog cerebellum during spontaneous and thyroxine-induced metamorphosis using the Golgi-Kopsch method and electron microscopy. Cells that possessed axosomatic synapses and resembled stellate cells were present even in the incipient molecular layer of the cerebellum in the premetamorphic tadpole. These cells may have originated from the early, transient wave of external granule cells that have been reported in the cerebellum of premetamorphic tadpoles in the first 6 months of development, and may constitute the variant population of stellate cells that are present later during development or the degenerating cells that have been observed during metamorphosis as scattered dying cells in the molecular layer. Typical stellate cells, whose development resembled the genesis and differentiation of stellate cells in birds and mammals, were initially observed at the outer border of the molecular layer that is adjacent to the external granular layer during the onset of metamorphosis. These stellate cells were bipolar with processes extending in a plane perpendicular to elongating parallel fibers, and with progressive development, became multipolar with dendrites oriented in various directions with respect to the pia. Stellate cell axons innervate the dendrites and somata of Purkinje cells and other stellate cells, and can be categorized into two types: (1) axons with extensive branching near the soma of origin, and (2) long axons with few branches that occasionally terminate in the Purkinje cell layer. Atypical neurons that did not resemble typical stellate cells were also present in the molecular layer; these might be classified as a stellate cell variant. The generation and differentiation of stellate cells can be induced 1 to 2 years prematurely by administering thyroid hormone to premetamorphic tadpoles. Like most events of cerebellar histogenesis in the frog, stellate cell development also appears to be largely a thyroid-dependent phenomenon.

Immunocytochemistry of luteinizing hormone-releasing hormone during spontaneous and thyroxine-induced metamorphosis of bullfrogs.

Double-bridge peroxidase-antiperoxidase immunocytochemistry was used to compare the developmental appearance of immunoreactive LH-RH (ir-LH-RH) in brains of bullfrog (Rana catesbeiana) tadpoles during either spontaneous or thyroxine-induced metamorphosis. During spontaneous metamorphosis, ir-LH-RH was localized in fibers of the external layer of the median eminence (ME) of stage XIII-XXV animals, while immunoreactive perikarya and other immunostained brain structures were absent. The extent and intensity of ME immunostaining increased concomitantly with measured ME morphological development. Tadpoles induced with thyroxine to metamorphic stages XIX-XXI exhibited ME structural development and neurohypophysial neurosecretory staining similar to spontaneously metamorphosed individuals of equal stages. However, comparable ME ir-LH-RH immunostaining and gonadal size were both less developed in thyroxine-treated animals, although increased relative to non-metamorphic vehicle-injected controls. These results indicate that the hypothalamic LH-RH system changes concurrently with ME structural development during spontaneous metamorphosis. Reduced ME ir-LH-RH staining and gonadal size in thyroxine-treated animals suggest that during prometamorphosis, factors other than thyroxine alone may coordinate the normal maturation of the hypothalamo-pituitary-gonadal axis of the bullfrog.

Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis

Purkinje cell maturation during thyroxine-induced metamorphosis in premetamorphic bullfrog tadpoles was studied using electron microscopy and Golgi (silver-impregnated) preparations. Cerebella from tadpoles were examined following 1, 2, or 3 weeks of thyroxine treatment. Particular attention was paid to possible differences between the two populations of Purkinje cells previously described, i.e. (i) the smaller population located in the dorsal part of the cerebellum, where the Purkinje cells show dendritic arborization long before the appearance of the external granular layer, and (ii) the larger population located in the middle and ventral regions of the cerebellum, where the Purkinje cells begin to undergo maturation during metamorphosis when the external granular layer is established. Following thyroxine treatment, both populations of Purkinje cells showed rapid maturational change. In the mature (dorsal) group, dendritic growth resumed in the presence of an external granular layer increasing the complexity of their dendritic arbors. Moreover, climbing fiber synapses translocated from contacts on the soma to the thorns of growing dendrites, and somatic processes often disappeared. The immature (ventral) group showed dramatic differentiation of the perikaryon including polarization of cytoplasm with subsequent dendritic outgrowth and formation of somatic processes in the presence of climbing fibers. Stellate cell contacts appeared on the smooth portion of the soma of many Purkinje cells. Dendritic growth during thyroxine-induced metamorphosis was characterized by growth (elongation) with minimal branching, which is initially observed during spontaneous metamorphosis. Typically, these growing dendrites ended in growth cones, some with one or several filopodia. Developing Purkinje cell dendritic spines formed synapses with parallel fibers. The present study has provided an example of the dramatic nature of thyroxine's action in inducing the complex series of detailed maturational changes in the cerebellum 1–2yr ahead of schedule. In addition, the results show that thyroxine-induced Purkinje cell maturation is more rapid and synchronous than that seen during spontaneous metamorphosis. It is concluded that Purkinje cell maturation during metamorphosis is largely dependent on thyroid hormone.

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis.

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.

Ultrastructural studies on Purkinje cell maturation in the cerebellum of the frog tadpole during spontaneous and thyroxine-induced metamorphosis.

The maturation of Purkinje cells in the cerebella of both thyroxine (T4)-induced and normally metamorphosing tadpoles was studied by transmission electron microscopy, with particular reference to the perikaryal changes. During the latter part of the prometamorphic phase, many Purkinje cells showed hypertrophied apical cones filled with mitochondria, Golgi elements and rosettes of ribosomes. In early metamorphic climax, the perikaryal cytoplasm displayed stratification, with an inner zone of perinuclear Nissl bodies and an outer region of neurotubules. At the onset of metamorphic climax, there was an abrupt appearance of numerous somatic processes, as well as climbing fiber boutons which synapsed with them on some cells. However, many Purkinje cells did not display somatic processes. Stellate cell synapses also were seen in considerable numbers. As metamorphic climax progressed to completion, the somatic processes steadily grew scarce with a concomitant increase in climbing fiber synapses on the major dendrites. Glial ensheathment of the Purkinje cell soma was also rapidly accomplished during metamorphic climax. In addition, premetamorphic bullfrog tadpoles were induced to metamorphose prematurely following treatment with T4 and then compared to normally metamorphosing tadpoles. Following 3 weeks of T4 treatment, large numbers of Purkinje cells displayed somatic processes. These processes were observed to be postsynaptic to climbing fibers and similar to those seen normally. Additionally, Purkinje cell hypertrophied apical cones were observed in treated tadpoles. These observations indicate that some aspects of Purkinje cell maturation during metamorphosis, especially the interaction of climbing fibers and somatic contacts, are T4-dependent. In both normal and induced metamorphosis, changes in frog Purkinje cells thus proceed at a tempo comparable to that of such gross morphological transformation as hindlimb growth.

Developmental changes in non-histone chromosomal proteins of bullfrog ( Rana catesbeiana)

1. 1. Chromatins were isolated from liver, brain, kidney, spleen, heart muscle and erythrocytes of the bullfrog and its tadpole, Rana catesbeiana and their non-histone chromosomal proteins (NHC proteins) were compared by a simple electrophoretic method. 2. 2. No qualitative but quantitative variations in the composition of NHC proteins were found among the tissues examined. 3. 3. The tissues of tadpole and frog were classified into three groups according to their electrophoretic pattern of NHC proteins; brain, heart muscle and spleen showing almost the same pattern, liver and kidney showing different pattern in some components, and erythrocytes showing markedly different pattern. 4. 4. Component D3 occurring in the frog liver, not in the tadpole, was found to increase in the tadpole liver during natural and thyroxine-induced metamorphosis.

Retardation of thyroxine-induced metamorphosis by Amphenone B in toad tadpoles.

The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.

Are surface potentials necessary for amphibian limb regeneration?

Surface potentials of amphibian limbs have been recorded during metamorphosis, skin shedding, and regeneration. Surface potentials are typical of adults; they appear at natural metamorphosis of Pleurodeles larvae and at thyroxine-induced metamorphosis of axolotl larvae. In adults during intermolt, the limbs are distally positive but during shedding of the stratum corneum surface potentials either disappear or the limb becomes distally negative. These variations are explained by modifications of the passive permeability of the epidermis. During limb regeneration, surface potentials show variations in magnitude and polarity but these variations are identical when skin wound healing alone occurs. For this reason and others, it is concluded that surface potentials are entirely epidermal and do not control limb regeneration.

Thyroxine stimulation of tadpole liver histone phosphorylation in vivo

The in vivo phosphorylation of histones in the livers of Rana catesbeiana tadpoles was followed during the course of thyroxine-induced metamorphosis. Phosphorylation of histones H1 and H2a, and possibly of histone H4 at a low level, was observed in all animals. After correction for specific radioactivity of liver inorganic phosphate pools, an apparent wave of phosphorylation of histones was found to occur between 2 and 8 days of thyroxine treatment, with a peak increase of approximately 2- to 5-fold for histones H2a and H1. The increases in liver histone phosphorylation are approximately coincident with well-documented increases in the levels of various liver enzymes and occur in the absence of any change in the low basal rate of histone or DNA synthesis in this organ. This is apparently the first instance of increased phosphorylation of both H1 and H2a which is not coincident with or precedent to increases in cellular proliferation rates.

Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis.

The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. gamma-Glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.

Comparative study of opiate and enkephalin receptors on lower vertebrates and higher vertebrates

1. The presence of opiate (μ) and enkephalin (δ) receptors have been characterized in the most primitive amphibians (Urodeles). 2. The ratio of the two types of receptors (δ:μ) is the same for Pleurodeles and Axolotl, but is lower than the ratio found in rat brain. 3. Thyroxine-induced metamorphosis of the Axolotl does not affect the presence and the number of the major population of “opiate” receptors, the μ receptors.

Histochemical patterns in the tadpole tail during normal and thyroxine-induced metamorphosis II. Succinic dehydrogenase, Mg- and Ca-adenosine triphosphatases, thiamine pyrophosphatase, and 5′-nucleotidase

The distributions of succinic dehydrogenase (SDH), adenosine triphosphatases (Mg-ATPase, Ca-ATPase), thiamine pyrophosphatase (TPPase), and 5′-nucleotidase (5′-Nase) were demonstrated histochemically in sections of growing and resorbing tails of Rana pipiens tadpoles. The main site of SDH activity was striated muscle, especially under the sarcolemma of small, peripheral cells. Mg-ATPase (pH 7.2) was similarly located but together with TPPase and 5′-Nase, was also present in the epidermis, blood vessel walls, and connective tissue, particularly the myocommata and outer notochordal sheath. TPPase was less active, and 5′-Nase still less active, than Mg-ATPase in the latter two locations. (Mg-ATPase and TPPase were also found in the ependyma of the spinal cord.) On the other hand, Ca-ATPase activity (pH 9.4) was almost uniformly distributed within all striated muscle cells; some activity was also evident in the epidermis and blood vessel walls, as well as in the connective tissue of the fins. In general, as the tail shortened during normal and thyroxine-induced metamorphosis, activity of SDH and of Mg-ATPase in peripheral muscle cells decreased, Ca-ATPase activity in muscle remained more or less the same, and activity of enzymes in other tissues of the tail either remained similar to increased. The present data on the differential distribution of five enzymes related to energy metabolism, transport, and muscle contraction, extend the histochemical catalog of enzyme localizations in the tail of tadpoles throughout early larval and metamorphic stages of development.

Inhibition of thyroxine-induced metamorphosis by prolactin in tadpoles, Rana catesbeiana.

Thyroxine (T4)-prolactin interactions on hepatic arginase and ornithine transcarbamylase (OTC) as well as hind legs, tail, digestive tract and median eminence were investigated in tadpoles, Rana catesbeiana. Prolactin completely blocked T4-induced tail resorption, but failed to suppress hind-leg growth, shortening of digestive tract and promotion by T4 of the median eminence development. Prolactin blocked T4-induced increase in hepatic arginase activity but not in hepatic OTC activity. A possibility that T4 and prolactin are regulating the hepatic arginase indirectly is discussed.

Antipain inhibits thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver.

The increased activity of carbamyl phosphate synthetase I [carbamoyl-phosphate synthase (ammonia); ATP: carbamate phosphotransferase (diphosphorylating), EC 2.7.2.5] in tadpole liver observed during thyroxine-induced metamorphosis was markedly inhibited by intraperitoneal injection of the microbial protease inhibitor antipain (0.1 micrometermol/g of body weight, twice daily). A somewhat less than maximal inhibition was seen when antipain was given only during the first 2 days of thyroxine treatment. On the other hand, little inhibition was observed when the inhibitor was given after the third or fourth day of thyroxine treatment. Antipain also inhibited thyroxine-induced increases of ornithine transcarbamylase (EC 2.1.3.3), arginase (EC 3.5.3.1), and succinate-cytochrome c reductase (EC 1.3.99.1) activities. Among other microbial protease inhibitors tested, chymostatin was nearly as effective as antipain, leupeptin was less effective, and pepstatin was ineffective. Analysis of the total liver protein and of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the inhibition was due to decreased amount of the enzyme protein. Antipain had no significant effect on leucine incorporation into total protein of tadpole liver. These results indicate the involvement of a proteolytic step in the pretranscriptional events in thyroxine-stimulated enzyme induction.