ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles

In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. We demonstrate that the MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, we identified the network of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, we show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. These observations lead us to propose a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi.     

Alanine aminopeptidase activity and autolysis in the tails of Rana catesbeiana larvae during metamorphosis.

1. Alanine aminopeptidase activity and autolysis increase concomitantly in tail tissue of Rana catesbeiana tadpoles during metamorphosis. 2. significant increases first appear at Taylor and Kollros state XX and coincide with the beginning of tail regression as determined by the tail wt body wt ration. 3. The results suggest a role of alanine aminopeptidase in the mechanism of tail resorption.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development

Numerous coactivators that bind nuclear hormone receptors have been isolated and characterized in vitro. Relatively few studies have addressed the developmental roles of these cofactors in vivo. By using the total dependence of amphibian metamorphosis on thyroid hormone (T3) as a model, we have investigated the role of steroid receptor coactivator 3 (SRC3) in gene activation by thyroid hormone receptor (TR) in vivo. First, expression analysis showed that SRC3 was expressed in all tadpole organs analyzed. In addition, during natural as well as T3-induced metamorphosis, SRC3 was up-regulated in both the tail and intestine, two organs that undergo extensive transformations during metamorphosis and the focus of the current study. We then performed chromatin immunoprecipitation assays to investigate whether SRC3 is recruited to endogenous T3 target genes in vivo in developing tadpoles. Surprisingly, we found that SRC3 was recruited in a gene- and tissue-dependent manner to target genes by TR, both upon T3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T3-dependent manner to the target TRbetaA promoter, suggesting either no recruitment or constitutive association. Finally, by using transgenic tadpoles expressing a dominant negative SRC3 (F-dnSRC3), we demonstrated that F-dnSRC3 was recruited in a T3-dependent manner in both the intestine and tail, blocking the recruitment of endogenous coactivators and histone acetylation. These results suggest that SRC3 is utilized in a gene- and tissue-specific manner by TR during development.     

Influence of Melatonin on the Rate of Rana pipiens Tadpole Metamorphosis In Vivo and Regression of Thyroxine-Treated Tail Tips In Vitro

Metamorphosis of Rana pipiens tadpoles may be retarded when the light phase of the light/dark (LD) cycle is shortened or when thyroxine (T₄) is given in the dark because melatonin peaks during the dark. Injection of premetamorphic tadpoles in spontaneous metamorphosis with melatonin (15 μg) retarded tail growth and hindlimb development on 18L:6D but had no significant effect on 6L:18D. During induced metamorphosis (30 μg/liter T₄), melatonin injections retarded tail resorption on 18L:6D and accelerated it on 6L:18D, but did not affect the hindlimb. When melatonin was injected during T₄ immersion at different times in the photophase on 18L:6D (L onset 0800 hr), tail regression was retarded by melatonin at 1430 or 2030 hr. At 0830 hr, shrinkage of tail length was accelerated whereas tail height was not affected. Tail tips in vitro induced to resorb by 0.2 μg/ml T₄ in Niu-Twitty solution regressed more slowly in the presence of melatonin (10 or 15 μg/ml) than with T₄ alone on both 6L:18D and 18L:6D. The findings implicate melatonin in LD cycle effects on tadpole metamorphic rate in vivo , show the importance of the time of melatonin injections, and indicate that melatonin antagonizes the metamorphic action of T₄ at the tissue level.     

Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus (Anura,Rhacophoridae)

Activities of acid phosphatase (normal and Co²⁺-sensitive), superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity (normal and Co²⁺-sensitive), and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis.     

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro.

The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.     

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10–500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.     

The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis

1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of (32)P into the different phospholipid constituents of microsomal membranes. 7. Nascent (14)C-labelled protein with the highest specific activity was recovered in the ;heavy' rough membrane fraction of microsomes, whereas little (14)C was associated with ;free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7-8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.     

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.     

Superoxide radical production in plasma membrane samples from regressing rat corpora lutea

Plasma membrane samples prepared from regressing rat corpora lutea were examined for production of the superoxide radical. A procedure was developed to purify membrane samples that were enriched approximately 15-fold with the plasma membrane marker enzyme, and superoxide radical levels were determined using electron spin resonance to measure Tiron semiquinone. During prostaglandin F2 alpha-induced and spontaneous regression, there was a significant increase in formation of superoxide radical that was not observed in plasma membrane samples from nonregressing corpora lutea. Plasma membrane incubation experiments indicated that the increase in production was temperature sensitive and reduced with inhibitors of phospholipase A2 and cyclooxygenase. Addition of superoxide dismutase or vitamin E abolished superoxide radical formation in vitro. Following the rise in superoxide radical levels during regression, there was also a significant decrease in the activity of the plasma membrane enzyme, Na+-K+ ATPase. These results indicate that the production of superoxide radical increases in plasma membrane samples prepared from regressing rat corpora lutea and that this increase is mainly due to the products of phospholipase A2 and cyclooxygenase activity.     

Mechanisms and Consequences of Developmental Acceleration in Tadpoles Responding to Pond Drying

Many amphibian species exploit temporary or even ephemeral aquatic habitats for reproduction by maximising larval growth under benign conditions but accelerating development to rapidly undergo metamorphosis when at risk of desiccation from pond drying. Here we determine mechanisms enabling developmental acceleration in response to decreased water levels in western spadefoot toad tadpoles (Pelobates cultripes), a species with long larval periods and large size at metamorphosis but with a high degree of developmental plasticity. We found that P. cultripes tadpoles can shorten their larval period by an average of 30% in response to reduced water levels. We show that such developmental acceleration was achieved via increased endogenous levels of corticosterone and thyroid hormone, which act synergistically to achieve metamorphosis, and also by increased expression of the thyroid hormone receptor TRΒ, which increases tissue sensitivity and responsivity to thyroid hormone. However, developmental acceleration had morphological and physiological consequences. In addition to resulting in smaller juveniles with proportionately shorter limbs, tadpoles exposed to decreased water levels incurred oxidative stress, indicated by increased activity of the antioxidant enzymes catalase, superoxide dismutase, and gluthatione peroxidase. Such increases were apparently sufficient to neutralise the oxidative damage caused by presumed increased metabolic activity. Thus, developmental acceleration allows spadefoot toad tadpoles to evade drying ponds, but it comes at the expense of reduced size at metamorphosis and increased oxidative stress.     

Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).     

Tail regression in Ciona intestinalis (Prochordate) involves a Caspase-dependent apoptosis event associated with ERK activation

Two apoptotic events take place during embryonic development of Ciona intestinalis. The first concerns extra-embryonic cells and precedes hatching. The second controls tail regression at metamorphosis, occurs through a polarized wave originating from tail extremity, and is caspase dependent. This was shown by: (1) in vivo incorporation of a fluorescent marker of caspase activation in different cell types of the tail; (2) detection of an activated form of caspase 3-like protein by western blotting; and (3) failure of 30% of larvae to undergo metamorphosis after treatment of fertilized eggs with a pan-caspase inhibitor. In addition, Ciona embryos express a single ERK protein, specifically phosphorylated at metamorphosis. ERK activation was shown to be located in cells of the tail. Addition of MEK inhibitor in the culture medium prevented ERK activation and metamorphosis. In silico analysis of Ciona genome pointed to 15 caspases with high homology with humans, and a single ERK gene with high homology to both mammalian ERK1 and ERK2. It is concluded that the sequence of events leading to metamorphosis includes ERK phosphorylation followed by caspase-dependent apoptosis and tail regression.     

Immunolocalization of Fibronectin and Laminin in the Amphibian Intestine During Spontaneous and Triiodothyronine-Induced Metamorphosis

Fibronectin and laminin were detected by indirect immunofluorescence in the intestine of Alytes obstetricans (anuran amphibian) during triiodothyronine (T3)-induced metamorphosis and spontaneous post-embryonic development. Fibronectin was first detected between a small number of connective tissue cells. As T3-treatment and spontaneous development progressed, fibronectin became detectable as a fine network extending throughout the whole thickness of the connective tissue and particularly in the core of the developing epithelial folds. During the first week of T3-treatment and throughout the spontaneous larval period, laminin was present as a linear band within the basement membrane. Between day 6 and 12 of hormonal treatment, an increase in the laminin fluorescent staining was noted. After hormonal treatment for two weeks and at the end of spontaneous metamorphosis, laminin staining was localized within the basement membrane of the folded epithelium and around muscle fibers. These observations indicate that variations in the density and distribution of extracellular matrix molecules are closely related spatiotemporarily to the structural changes occurring in the connective and muscle tissues of the intestine during metamorphosis.