Xenopus Metamorphosis Research Articles

Activity and expression of Xenopus laevis matrix metalloproteinases: Identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals

AbstractProlactin (PRL) has long been implicated in Xenopus metamorphosis as an anti‐metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T3). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti‐TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase‐3 (XCL3) and collagenase‐4 (XCL4) expression in a cultured Xenopus laevis cell line, XL‐177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T3‐treated cells, but were differentially induced by PRL in a dose‐ and time‐dependent fashion. Moreover, treatment with IL‐1α as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL‐induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL‐induced collagenase expression. Interestingly, XCL3 expression could be induced in XL‐177 cells by T3, but only when co‐cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T3‐induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T3 and further suggest that PRL is a candidate regulator of TH‐independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development. J. Cell. Physiol. 201: 165, 2004. © 2004 Wiley‐Liss, Inc.

Activity and expression of Xenopus laevis matrix metalloproteinases: identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals.

Prolactin (PRL) has long been implicated in Xenopus metamorphosis as an anti-metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T(3)). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti-TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase-3 (XCL3) and collagenase-4 (XCL4) expression in a cultured Xenopus laevis cell line, XL-177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T(3)-treated cells, but were differentially induced by PRL in a dose- and time-dependent fashion. Moreover, treatment with IL-1alpha as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL-induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL-induced collagenase expression. Interestingly, XCL3 expression could be induced in XL-177 cells by T(3), but only when co-cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T(3)-induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T(3) and further suggest that PRL is a candidate regulator of TH-independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development.

Regulation of Specific Developmental Fates of Larval- and Adult-Type Muscles during Metamorphosis of the Frog Xenopus

During anuran metamorphosis, larval-type myotubes in both trunk and tail are removed by apoptosis, and only trunk muscles are replaced by newly formed adult-type myotubes. In the present study, we clarified the regulatory mechanisms for specific developmental fates of adult and larval muscles. Two distinct (adult and larval) types of myoblasts were found to exist in the trunk, but no or very few adult myoblasts were found in the tail. Each type of myoblast responded differently to metamorphic trigger, 3,3′,5-triiodo- l-thyronine (T 3) in vitro. T 3-induced cell death was observed in larval myoblasts but not in adult myoblasts. These results suggest that the fates (life or death) of trunk and tail muscles are determined primarily by the differential distribution of adult myoblasts within the muscles. However, a transplantation study clarified that each larval and adult myoblast was not committed to fuse into particular myotube types, and they could form heterokaryon myotubes in vivo. Cell culture experiments suggested that the following two mechanisms are involved in the specification of myotube fate: (1) Heterokaryon myotubes could escape T 3-induced death only when the proportion of adult nuclei number was higher than 70% in the myotubes. Apoptosis was not observed in any larval nuclei within the surviving heterokaryon myotubes, suggesting the conversion of larval nuclei fate. (2) Differentiation of adult myoblasts was promoted by the factor(s) released from larval myoblasts in a cell type-specific manner. Taken together, the developmental fate of myotubes is determined by the ratio of nuclei types, and the formation of adult nuclei-rich myotubes was specifically enhanced by larval myoblast factor(s).

Thyroid hormone regulation of a transcriptional coactivator in Xenopus laevis: Implication for a role in postembryonic tissue remodeling

Thyroid hormone (TH) affects biological processes by regulating gene transcription through TH receptors (TRs). In the presence of TH, TR activates target gene transcription by recruiting one or more transcription coactivators belonging to diverse groups. Here, we demonstrate that during TH-dependent anuran metamorphosis, one such coactivator gene, the Xenopus laevis homolog of human Trip7, is up-regulated by TH. Kinetic studies suggest that Xenopus Trip7 is most likely induced indirectly by TH in a tissue-dependent manner. In the intestine, which undergoes extensive remodeling as the animal changes from being herbivorous to carnivorous, Trip7 is expressed at high levels during but not before or after metamorphosis. It is also up-regulated in other growing or remodeling tissues such as the brain and limb but not in degenerating tadpole tail skin. By using frog oocyte as a model, we show that Trip7 influences basal transcription in a chromatin structure-dependent manner but enhances the function of liganded TR regardless of the chromatin structure of the target promoter. In vitro studies indicate that Trip7 interacts directly with TR. These results suggest that during Xenopus metamorphosis, TH up-regulates, albeit indirectly, Trip7 to enhance TR function during larval-to-adult tissue transformation.

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.

Conversion of red blood cells (RBCs) from the larval to the adult type during metamorphosis in Xenopus: specific removal of mature larval-type RBCs by apoptosis.

The conversion of hemoglobins (Hbs) and red blood cells (RBCs) from the larval to the adult type was monitored during normal metamorphosis in Xenopus laevis, and in artificially induced metamorphosis-arrested and precociously metamorphosed animals by means of SDS-PAGE, Hb immunohistochemistry, and double-staining with in situ DNA nick-end labeling (TUNEL) for detection of apoptosis and Hb immunostain. During normal metamorphosis, larval RBCs gradually decreased and, conversely, adult RBCs increased in number. However, in metamorphosis-arrested tadpoles, the larval-adult conversion of RBCs did not occur within 4 weeks, but did rather within 6 months after the controls metamorphosed. In order to identify possible mechanisms for the specific removal of larval RBCs from circulation in metamorphosing and metamorphosed animals, double-staining experiments with TUNEL and Hb immunostain were carried out. During metamorphic climax, many larval RBCs expressed TUNEL-positive reactions in the spleen, suggesting that the larval RBCs were specifically removed from the spleen during metamorphosis. When the larval RBCs were transferred to the circulatory system of histocompatible control adults, they survived for a long time, and no transferred RBCs showed TUNEL-positive reactions. In contrast, larval RBCs transferred to histocompatible adults that had been treated with T3 were reduced in number in the circulatory system of the recipients. Double-staining experiments demonstrated that the transferred larval RBCs underwent apoptosis in the spleen and liver of the adult recipients treated with T3, indicating that the mature larval-type RBCs were specifically removed from metamorphosing animals by apoptotic cell death under the influence of THs.

Thyroid hormone regulation of apoptotic tissue remodeling: Implications from molecular analysis of amphibian metamorphosis

Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de novo development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process.

Effects of pond water, sediment, and sediment extracts from minnesota and vermont, USA, on early development and metamorphosis of xenopus.

In recent studies, a high incidence of amphibian mortality and malformation has been reported in the field, suggesting that toxic and/or bioactive agents are present in the environment of the affected amphibians. This study provides evidence for this hypothesis, because it applies to several affected ponds in Minnesota and Vermont, USA. Three developmental bioassays were carried out on samples from three reference and three test sites in Minnesota and one reference and three test sites, in Vermont. The bioassays utilized Xenopus as a model system, measuring altered developmental patterns during the first 4 d of development (frog embryo teratogenesis assay-Xenopus [FETAX]), hind-limb development over a 30-d period, and tail length resorption over a 14-d period. Strong correlations were observed among the results for all three in vitro bioassays, as well as between adverse developmental effects in vitro and in the field.

Isolation and Characterization of NUC70, a Cytoplasmic, Hematopoietic Apoptotic Endonuclease

Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and active over a pH range of 6-8. When incubated with isolated HeLa nuclei, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic and nuclear endonuclease activities during apoptosis show that NUC70 is a cytoplasmic endonuclease that is translocated to the nucleus after the initiation of apoptosis. We confirmed this with immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.

3,5,3'-Triiodo-L-thyronine potentiates all-trans-retinoic acid-induced apoptosis during differentiation of the promyeloleukemic cell HL-60.

Although the programmed cell death mediated by thyroid hormone is not well evaluated in mammalian cells, thyroid hormone plays a crucial role in differentiation of the cells during the metamorphosis of Xenopus, suggesting that thyroid hormone has the potential ability to induce the apoptosis. To investigate the thyroid hormone-inducible apoptosis, we cultured HL-60 cells with various amounts of all-transretinoic acid (RA) and L-T3. T3 alone did not induce the apoptosis of the cells. T3, however, suppressed the proliferation of cells in the presence of RA. DNA ladder and microscopical examination showed that the reduction of cell number was due to the apoptosis induced by RA. These findings suggested that T3 affects the apoptotic process during the differentiation of HL-60 cells by RA. T3-inducible apoptosis may require the factors augmented by RA in HL-60 cells.

Thyroid Hormone Controls the Onset of Androgen Sensitivity in the Developing Larynx ofXenopus laevis

Gonadal differentiation, the onset of androgen-stimulated laryngeal growth and the genesis of a sex difference in laryngeal innervation, all temporally coincide with thyroid hormone (TH)-induced metamorphosis inXenopus laevis.To explore the role TH plays in the ontogeny of theXenopusandrogen-sensitive vocal neuromuscular system, we examined gonadal and laryngeal development in tadpoles in which metamorphosis had been blocked by treatment with the thyroxine synthesis inhibitor propylthiouracil (PTU). PTU treatment did not arrest gonadal differentiation. Testes from PTU-treated male tadpoles had seminiferous tubules and advanced stage male germ cells, while in females stage 1 oocytes were present. In contrast to the gonads, PTU did block morphological development of the larynx. Tadpoles treated with PTU for 50 or 100 days had larynges which structurally resembled those of stage 54 control tadpoles. PTU-treated animals did not exhibit the extensive development of the laryngeal cartilage seen in untreated animals. Laryngeal cartilages of hypothyroid tadpoles exhibited low density and minimal patterning of chondrocytes; the complex lumen and marked expansion of the dilator muscles characteristic of 50- and 100-day untreated animals were absent. Laryngeal growth evoked by exposure to exogenous androgen (dihydrotestosterone) was entirely prevented by PTU treatment. Hypothyroid tadpoles did not exhibit the decline in laryngeal nerve axon number characteristic of age-matched controls, nor were laryngeal nerve axon numbers sexually dimorphic. PTU treatment also interfered with the myelination of laryngeal axons. We conclude that while gonadal differentiation is independent of TH, androgen sensitive laryngeal development and sexually dimorphic laryngeal innervation require exposure to secreted TH.

Transient expression of stromelysin-3 mRNA in the amphibian small intestine during metamorphosis.

It has been suggested that a matrix metalloproteinase, stromelysin-3 (ST3), is an important enzyme for epithelial transformation; the ST3 gene is known to be regulated by thyroid hormone during Xenopus metamorphosis. In this study, we have examined the distribution of ST3 mRNA in the small intestine of Xenopus during metamorphosis by using in situ hybridization. Around stage 58, ST3 mRNA is first detectable in larval fibroblasts near the muscular layer, and then increases in amount throughout the entire region of connective tissue. By stage 61, when connective tissue cells are rapidly increasing in number, ST3 mRNA is localized in fibroblasts just beneath the epithelium. This localization of ST3 mRNA is in good temporal accordance with modification of the basement membrane and epithelial transformation from the larval to adult form. Thereafter, ST3 mRNA gradually decreases and is no longer detected after stage 63 when the adult epithelium has completely replaced the larval type. The transient expression of ST3 mRNA in the fibroblasts of connective tissue during metamorphosis indicates that it plays an important role in epithelial transformation by changing the basement membrane.

Cell-specific and spatio-temporal expression of intestinal fatty acid-binding protein gene during amphibian metamorphosis.

Intestinal fatty acid-binding protein (IFABP) gene is known to be regulated during Xenopus metamorphosis. To determine the relationship between its regulation and cellular differentiation during metamorphosis, we have examined the distribution of IFABP mRNA in the Xenopus digestive tract by in situ hybridization techniques. Throughout all stages examined, transcripts of IFABP gene were observed exclusively in absorptive epithelial cells of the small intestine, and they decreased in amount towards the posterior intestine. Around stage 58, just before metamorphic climax, IFABP mRNA level began to decrease in larval absorptive cells that still remained intact morphologically. Thereafter, IFABP mRNA was no longer detected among larval cells. In turn, at stage 62, IFABP mRNA became detectable in some of the newly formed adult epithelium that had not yet developed a brush border, but not in the remaining larval cells. By the end of metamorphosis, IFABP mRNA became more abundant towards the crest of intestinal folds. These results suggest that IFABP gene expression is specific for absorptive epithelial cells of the small intestine and is regionally regulated along the intestinal anterior-posterior axis in both tadpoles and frogs and also along the trough-crest axis of frog intestinal folds. In addition, our present study directly shows that IFABP mRNA level decreases in larval absorptive cells but increases in adult ones during metamorphosis, preceding morphological changes of both types of cells. Therefore, the regulation of IFABP gene is an early event during both larval epithelial cell death and adult epithelial cell differentiation.

Expression of Magainin Antimicrobial Peptide Genes in the Developing Granular Glands of Xenopus Skin and Induction by Thyroid Hormone

The granular gland of amphibian skin is an epithelial derivative that first appears during metamorphosis. The granular glands of Xenopus laevis are an abundant source of magainins, a family of peptides with antimicrobial activity in vitro. We demonstrate the developmental expression and thyroid hormone induction of magainin and PGLa (peptide with aminoterminal glycine and carboxyterminal leucinamide), the two most abundant members of the magainin peptide family. Magainin and PGLa mRNA levels increase dramatically over the 2-day period between stage 58 and stage 60 and remain high during the remainder of metamorphosis. We used in situ hybridization to localize magainin and PGLa mRNAs to the developing granular gland. Magainin and PGLa peptides are also first detected during metamorphosis, as demonstrated by extraction of active peptides from stage 60 tadpoles and immunogold histochemical localization of magainin and PGLa to the granules of the skin granular gland. We demonstrate the premature induction of magainin and PGLa expression upon administration of exogenous thyroid hormone to stage 54 tadpoles. Further investigation of the developmental expression and induction of the magainin peptides will provide insight into their possible biological functions during Xenopus metamorphosis.

Interplay between thyroid hormone and estrogen in modulating expression of their receptor and vitellogenin genes during Xenopus metamorphosis

Many postembryonic developmental processes are regulated by an intricate interplay among hormones and growth factors. Thyroid hormone (TH) and estrogen are well known to be individually and obligatorily required for the initiation and progression of amphibian metamorphosis and vitellogenesis. However, whether or not a possible interplay between these two hormones would affect these two developmental processes is not known. Here we report on how triiodothyronine (T 3) enhances the precocious activation of vitellogenin (Vit) genes by estradiol (E 2) in Xenopus tadpoles during metamorphosis. Using a combination of filter hybridization, RNase protection assay and in situ hybridization, we first show that very low doses (10 −9M) of exogenous T 3 will autoinduce thyroid hormone receptor (TR) mRNA in several tissues of premetamorphic tadpoles. The same treatment enhances and accelerates the precocious activation of the silent vitellogenin genes by E 2 at metamorphic climax (stages 60–64) but not before mid-metamorphosis (stages 56–58). This developmental stage dependency may be explained by our finding that, under the same experimental conditions, T 3 fails to alter the autoinduction of ER mRNA at mid-metamorphosis but strongly potentiates it at metamorphic climax. Thus a developmental stage specific interplay between thyroid hormone and estrogen determines the kinetics and extent of activation of vitellogenin and estrogen receptor genes during Xenopus postembryonic development.

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T 3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T 3 action we examined the effects of T 3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T 3 alone and T 3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.

Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus.

Abstract The developing thymus in Xenopus was examined at four different levels: 1) precursor immigration of cytogenetically distinct embryonic stem cells; 2) waves of colonization during tadpole life and metamorphosis; 3) inter-thymic exchange of cells between separate lobes; and 4) development of cortical and medullary thymocytes. Based on the flow cytometric analysis of cytogenetically distinct thymocytes, there were at least two periods of stem cell immigration into the thymus, one during early larval life and the second before or during metamorphosis. Within the thymus, cohorts of cells derived from the first wave of immigration expanded at different times. The initial expansion occurred before 35 days of development. Cells involved in the second period of expansion were also derived from the initial immigrants, expanded after 35 days, and resulted in a turnover of thymocytes during the larval period. Precursor cells entering the thymus during metamorphosis expanded and resulted in an additional replacement of thymocytes. Cortical and medullary thymocytes were isolated from animals that received embryonic stem cell grafts. No differences in the presence or absence, or in the percentages, of donor thymocytes in these different fractions were observed. When limiting numbers of stem cells were transplanted, several cases of asymmetrical thymic lobe colonization were observed. These data suggested that an inter-thymic exchange of cells did not occur during larval life.

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a “mature erythrocyte” antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of “mature erythrocyte” antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long period of time express intermediate levels of MHC antigens, suggesting a “leaky” expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.

Changes in the immune system during metamorphosis of Xenopus

Profound immunological changes occur as tadpoles metamorphose into adult amphibians. These include the expression of a different antibody repertoire, a lessening of skin graft tolerance, the appearance on leukocytes of class I MHC antigens. Here Martin Flajnik and his colleagues review what is known of these changes in Xenopus and speculate on how they may occur.

Oestrogen receptor levels and vitellogenin synthesis during development of Xenopus laevis.

The synthesis of vitellogenin, the precursor of the major eggyolk proteins, becomes inducible by oestrogen in Xenopus laevis larval liver during metamorphosis1–4, well before vitellogenin is required for oocyte growth. The synthesis in vivo of some liver proteins is responsive to oestradiol at earlier stages, but vitellogenin inducibility does not appear until a precise point during metamorphic climax, stage 62, when many ‘adult’ liver proteins first start to be synthesized5. This suggests that vitellogenin inducibility is not determined simply by the presence of an oestrogen receptor. The receptor identified and characterized in fully inducible adult liver6,7 (also detected by others8) is unusual because it is present at very low concentrations and 50% is found in the nucleus in unstimulated animals6,7, and is itself synthesized in response to oestrogen, raising the level in the nucleus9. Here we have measured nuclear receptor levels and the effect of oestrogen on these during Xenopus metamorphosis. Larvae which cannot be induced to synthesize vitellogenin contain an oestrogen receptor with the same affinity and specificity as the adult receptor. Treatment with oestrogen raises the level of nuclear oestrogen receptor in inducible but not in uninducible larvae. These and additional results from- thyrostatic larvae suggest that an increase in the level of nuclear receptor is necessary for the induction of vitellogenin synthesis, and that the onset of vitellogenin inducibility might be determined by the ability of oestrogen to increase nuclear receptor levels.