Roles of macrophages in programmed cell death and remodeling of tail and body muscle of Xenopus laevis during metamorphosis

 Examination was made of the involvement of macrophage phagocytosis in programmed cell death of tail and body muscle of the frog, Xenopus laevis, during metamorphosis by electron microscopy and immunohistochemical analysis. Electron microscopic observation revealed that macrophages were often found to be present in body and tail muscles at the most active stage of metamorphosis and to actively phagocytose apoptotic muscle fragments. Developmental changes in macrophages were examined using the macrophage-specific antibody, HAM56. Macrophages initially appeared in the early climax stage (stage 59), when the triiodothyronine (T₃) level was high, increased rapidly during the process of muscle cell death, and assumed their greatest number at the late climax stage (stage 63/64). They decreased after stage 65/66, with a decrease in T₃. Distribution and change in the number of macrophages were the same as those of muscle apoptotic bodies (sarcolytes) during metamorphosis, which suggests an interactive mechanism between macrophages and dying muscle cells. For clarification of this, study was made of the expression of HAM 56 antigens that were X. laevis homologs of mouse attachmin, non-specific adhesion proteins in macrophages. The expression of HAM56 antigens in macrophages was found to increase with macrophage phagocytosis at the late climax stage, thus, macrophage differentiation would appear to take place during metamorphosis and HAM56 antigens may be essential for macrophage–dying muscle cell interactions. Accepted: 29 May 1997     

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

Oxidative stress, tissue remodeling and regression during amphibian metamorphosis

Anuran metamorphosis is characterized by rapid and drastic changes in the body form and function under the influence of thyroid hormones. We evaluated the involvement of reactive oxygen species and antioxidant defenses during intestinal remodeling and tail regression of tadpoles of Xenopus laevis. Oxidative stress resulting from depletion in catalase and reduced glutathione, and simultaneous increase in lipid peroxidation during intestinal remodeling as well as tail regression are probably responsible for cell death and differentiation in these organs. Gene expression data for superoxide dismutase and catalase supports this contention. A dramatic increase in another antioxidant, ascorbic acid content of both these organs during metamorphic climax indicates its multifactor role such as collagen synthesis in intestine and controlled tail regression. These findings suggest that the cellular environment in the intestine and tail becomes progressively more oxidizing during its remodeling and regression respectively.     

Changes in corticosterone and aldosterone concentrations in various tissues of Xenopus laevis tadpoles during the metamorphosis

During the metamorphosis of Xenopus laevis tadpoles, tissue concentrations of corticosterone and aldosterone did not change significantly in forelegs and hindlegs; they increased in tail, liver, skin and intestine. The rise of corticosteroid concentrations appeared in tissues which were deeply transformed during the first part of the climax, when plasma levels of corticosteroids also increased. Highest tissue levels, attained at the mid-climax, were maintained at least until the end of metamorphosis although plasma concentrations of two steroids were then abruptly fallen. Tissues able to retain corticosteroids reacted as Vertebrate "target tissues" and transformations which took place in them could be dependent, at least partially, on corticosteroids.     

Thyroxine-binding proteins in liver and tail of Rana catebeiana undergoing metamorphosis.

Presence of a thyroxine-binding protein was demonstrated in vivo in cell sap of tail and liver of metamorphosing Rana catesbeiana tadpoles. Thyroxine-binding protein was not present in tail of prematamorphic tadpoles while it appeared during progressing metamorphosis roughly coinciding with the beginning of tail resorption. Susceptibility to pronase indicates that this thyroxine-binding macromolecule is protein in nature. Thyroxine-binding in liver was already present during premetamorphic stages and increased further during metamorphosis. A further difference between tail and liver thyroxine-binding protein was evidenced by molecular sieve chromatography on Sephadex G-200 indicating a molecular weight of thyroxine-binding protein in the tail of 60 000 as opposed to 42 000 for liver. Scatchard analysis of tail cell sap of tadpoles in metamorphic climax revealed a high affinity thyroxing binding site (Kd of 2 - 10(-10) M) of low capacity (1.7 pmol per mg protein) while tadpoles in premetamorphic stage had a thyroxine-binding site of lower affinity (9 - 10(-10) M) and higher capacity (4.8 pmol per mg protein). Thus affinity of thyroxine binding is 4-fold in metamorphic climax and appears to reflect the appearance of thyroxine binding observed in vivo.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren     

Programmed cell death in Xenopus laevis spinal cord, tail and other tissues, prior to, and during, metamorphosis

Programmed cell death is necessary for the shaping and remodelling of nervous and non-nervous tissues during development. Amphibia, whose body undergoes profound modifications during metamorphosis, are particularly useful models for studying the relationship between cell death in muscles and other non-nervous tissues on the one hand, and in the nervous system connected with these tissues on the other hand. We checked the occurrence of apoptotic cells (identified by TUNEL labelling) in different organs and regions from hatching (stages 35-36) to climax (stages 63-64) in the African Clawed Frog Xenopus laevis. Some organs (e.g., skin and digestive tract) contained apoptotic cells during the entire period studied. In transitory organs (cement gland and gills), a single wave of cell death occurred during the regression of these tissues. In order to compare the timing of cell death in the spinal cord with that of tail regression, we counted the number of TUNEL-positive cells in spinal cord sections taken from animals between stages 54 and 64. Three-dimensional reconstructions using confocal microscopy of vibratome slices immunostained for the detection of c-Jun-like protein accumulated in the cytoplasm of apoptotic cells showed numerous cells at various degrees of degeneration. Many of these cells still presented the morphological characteristics of neurones. The peak of apoptosis was found at stage 58, preceding tail regression. This suggests that neural cell death is not a consequence but rather an element upstream in the chain of events leading to tail degeneration.     

黑斑蛙变态过程中甲状腺发育及甲状腺激素分泌

系统研究了我国本土两栖动物种黑斑蛙（Rana nigromaculata）变态发育过程中甲状腺组织学和甲状腺激素水平的变化,为甲状腺生物学和甲状腺干扰研究提供基础数据。黑斑蛙蝌蚪发育的形态变化: 第26-40阶段,后腿芽生长并逐渐分化出五趾结构;42阶段,开始进入变态高峰期,前肢展开,尾吸收,蝌蚪身体发生巨大形变;46阶段,蝌蚪完全变态成小蛙。随着形态学的变化,甲状腺的组织结构也发生明显的变化: 26-37阶段,甲状腺体积较小,增长缓慢;38阶段甲状腺体积迅速膨大,进入高峰期,甲状腺的发育达到顶峰;随着变态完成,甲状腺又逐渐缩小。甲状腺组织学变化的同时,甲状腺激素水平也相应发生变化: 在变态前期,下颌中3,3',5-三碘代-L-甲腺原氨酸（T3）水平增长缓慢,进入变态期后,T3含量迅速升高,在变态高峰期达到峰值,随后下降。以上结果表明,黑斑蛙发育过程中甲状腺组织学的变化与甲状腺激素水平的波动相吻合。对黑斑蛙甲状腺系统的研究,可为日后使用黑斑蛙开展甲状腺干扰作用的研究提供基础。       

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.     

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis

 The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T₃ action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T₃ and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59–60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T₃ (10^-8 M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occured specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T₃ action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T₃ and 32 times as much by the action of T₃ plus HC, indicating the important action of T₃–HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T₃–HC synergism on adult erythroblast proliferation and T₃-mediated programmed death of larval precursor cells. Accepted: 14 January 1999     

Cell-cell and cell-ECM interactions in epithelial apoptosis and cell renewal during frog intestinal development

Amphibian intestinal remodeling during metamorphosis is a developmental system that is entirely controlled by thyroid hormone. It transforms a simple tubular organ into a complex multiply folded frog intestine similar to that in higher vertebrates. This process involves the degeneration of the larval epithelium through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. Earlier morphological and cellular studies have provided strong evidence implicating the importance of cell-cell and cell-ECM (extracellular matrix) interactions in this process. The recent molecular characterization of the genes that are regulated by thyroid hormone has begun to reveal some molecular clues underlying such interactions. In particular, theXenopus putative morphogen hedgehog appears to be involved in regulating/mediating cell-cell interactions during adult epithelial proliferation, differentiation, and/or intestinal morphogenesis. On the other hand, several matrix metalloproteinases (MMPs) may be involved in remodeling the ECM. Of special interest is stromelysin-3, whose spatial and temporal expression profile during intestinal metamorphosis implicates a role in ECM remodeling, which in turn facilitates cell fate determination, i.e., apoptosis vs proliferation and differentiation. Understanding the mechanisms of action for those extracellular molecules will present a future challenge in developmental research.     

Amphibian organ remodeling during metamorphosis: insight into thyroid hormone-induced apoptosis

During amphibian metamorphosis, the animal body dramatically remodels to adapt from the aquatic to the terrestrial life. Cell death of larval organs/tissues occurs massively in balance with proliferation of adult organs/tissues, to ensure survival of the individuals. Thus, amphibian metamorphosis provides a unique and valuable opportunity to study regulatory mechanisms of cell death. The advantage of this animal model is the absolute dependence of amphibian metamorphosis on thyroid hormone (TH). Since the 1990s, a number of TH response genes have been identified in several organs of Xenopus laevis tadpoles such as the tail and the intestine by subtractive hybridization and more recently by cDNA microarrays. Their expression and functional analyses, which are still ongoing, have shed light on molecular mechanisms of TH‐induced cell death during amphibian metamorphosis. In this review, I survey the recent progress of research in this field, focusing on the X. laevis intestine where apoptotic process is well characterized at the cellular level and can be easily manipulated in vitro. A growing body of evidence indicates that apoptosis during the intestinal remodeling occurs not only via a cell‐autonomous pathway but also via cell–cell and/or cell–extracellular matrix (ECM) interactions. Especially, stromelysin‐3, a matrix metalloproteinase, has been shown to alter cell–ECM interactions by cleaving a laminin receptor and induce apoptosis in the larval intestinal epithelium. Here, I emphasize the importance of TH‐induced multiple apoptotic pathways for massive and well‐organized apoptosis in the amphibian organs and discuss their conservation in the mammalian organs.     

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.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to 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).     

Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during <Emphasis Type="Italic">Xenopus</Emphasis> metamorphosis

Background

The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.

Results

Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.

Conclusions

Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

     

Developmental implications of differential effects of calcium in tail and body skin of Anuran tadpoles

The effects of external Ca(++) on metamorphosis of Rana catesbeiana tadpoles were assessed. Treatment of tadpoles with Ca(++) (0.05 mM) during early prometamorphic stages induced precocious metamorphic events such as tail regression, shortening of the intestine, forelimb emergence, and keratinization of body epidermis within 23 days of treatment compared to control tadpoles still in mid-prometamorphic stages. These effects of Ca(++) are probably mediated by the thyroid gland, as indicated by histological features of the gland at the light and electron microscopic levels. Calcium levels of tail and body skin were measured at various stages of development by atomic absorption spectrophotometry. In control and experimental groups, body skin had significantly higher Ca(++) concentrations than tail skin. There were no statistically significant effects of developmental stage on Ca(++) levels of tail or body skin. Experimental Ca(++) treatment significantly increased Ca(++) concentration in tail but not body skin. Ultrastructure studies and gel electrophoresis indicated that calcium induced keratinization of body skin, but not tail epidermis. Ca(++)-treated tail epidermis showed various autolysing figures in apoptotic cells. In summary, calcium treatment accelerated metamorphosis and induced the following region-dependent cellular events: keratinization of body skin-a characteristic of adult epidermis-and programmed cell death in the tail. Whatever signal elicited by calcium in this experimentally induced accelerated metamorphosis is probably mediated via the thyroid gland.     

Dermal breaks: migratory cell pathways opened in anuran tadpole skin at climactic metamorphosis

In this report, we studied the formation of breaks in the frog dermis during its remodelling at climactic metamorphosis. This remodelling consisted of detachment of the basement lamella collagen from the epidermis. The detached part, called derived collagen, was progressively fractured by breaks. We focused our attention on dermal cell localization during break formation. Firstly, at early climax, dermal cells were localized inside fractures opened in the derived collagen. Secondly, at the later climax, the fractures became breaks, making room for the dermal cells themselves. Thirdly, in derived collagen of the froglet, the well-opened breaks contained elongated dermal cells. At climax, DAB immunoperoxidase staining of fibronectin revealed a granular pattern at the surface of epidermal and dermal cells. Unexpected staining revealed that the dermal breaks contained fibronectin in the form of vertical lines. The foregoing results suggest that the dermal breaks are migratory pathways for dermal cells in derived collagen remodelled at climax.