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Thymus ontogeny in frogs: T-cell renewal at metamorphosis.

Metamorphosis in amphibians presents a unique problem for the developing immune system. Because tadpoles are free-living, they need an immune system to protect against potential pathogens. However, at metamorphosis, they acquire a variety of new adult-specific molecules to which the tadpole immune system must become tolerant. We hypothesized that Xenopus laevis tadpoles may avoid potentially destructive antiself responses by largely discarding the larval immune system at metamorphosis and acquiring a new one. By implanting triploid (3N) thymuses into diploid (2N) hosts, we examined the influx and expansion of host T-cell precursors in the donor thymus of normally metamorphosing and metamorphosis-inhibited frogs. We observed that donor thymocytes are replaced by host-derived cells during metamorphosis, but inhibition of metamorphosis does not prevent this exchange of cells. The implanted thymuses export T cells to the spleen. This donor-derived pool of cells declines after metamorphosis in normally developing frogs but is retained to a greater extent if metamorphosis is inhibited. These studies confirm previous observations of a metamorphosis-associated wave of expansion of T cells and demonstrate that it is not dependent on the relatively high concentrations of thyroid hormones required for metamorphosis. Although some larval T cells persist through metamorphosis, others may be destroyed or the larval population is significantly diluted by the expanding adult population.

Animals

Changes in the ventral dermis and development of iridophores in the anadromous sea lamprey, Petromyzon marinus, during metamorphosis: an ultrastructural study.

The ultrastructural changes that take place in the ventral dermis along with the development of iridophores were examined in the anadromous sea lamprey, Petromyzon marinus, during metamorphosis. There is a disruption of all components of the ventral dermis and a reformation that results in a structure very similar to that prior to metamorphosis. Although not a dermal component, a layer of iridophores develops directly beneath the dermis during late metamorphosis. The dermal endothelium is lost by mid metamorphosis (stage 4) and the highly organized collagenous lamellae making up the bulk of the dermis become disrupted by the migration of fibroblasts into the region. Many of these fibroblasts are involved in the degradation of the lamellae. By stage 5 of metamorphosis some fibroblasts become highly active collagen synthesizing cuboidal shaped cells that align to form a layer above the reformed dermal endothelium. New lamellae are formed by these cuboidal cells which then divide and migrate into the lamellae where they assume the characteristic attenuated appearance of fibroblasts in the adult dermal lamellae region. Iridophores first appear during stage 5 directly beneath the dermal endothelium. Reflecting platelets develop from double membraned vesicles associated with the Golgi apparatus. By late metamorphosis, stacks of trapezoidal shaped platelets fill the cytoplasm of the iridophores. The significance of the changes in the dermis during metamorphosis are discussed. This work is part of a continuing series of studies on the connective tissues in the anadromous sea lamprey.

Animals

Patterns of serotonin and SCP immunoreactivity during metamorphosis of the nervous system of the red abalone, Haliotis rufescens.

Larvae of the red abalone, Haliotis rufescens, rely on external chemical cues to trigger metamorphosis; thus, the timing of metamorphosis is dependent upon the larva's chance encounter with the appropriate substrate. We examined the effect of the timing of metamorphosis on the development of the central nervous system (CNS), concentrating on the pattern of serotonin and small cardioactive peptide- (SCP) immunopositive neurons in the cerebral ganglia. By 4 days postfertilization the cerebral ganglion has five pairs of serotonin-immunoreactive (IR) neurons, one pair of which (the V cells) innervate the velum. This complement of cells remains stable for as long as the larval stage persists but metamorphosis causes the rapid loss of the V cells. In the case of SCP-IR neurons, one pair is present prior to metamorphic competency, but as larvae continue to age in the absence of inducing cues, additional pairs are gradually added. Metamorphosis causes an acceleration in SCP-IR neuron addition. This separation of developmental patterns is well adapted for the inherent uncertainty of the timing of metamorphosis in abalone larvae.

Animals

Fatty metamorphosis in patients with jejunoileal bypass.

Two-thirds of all preoperative patients had some degree of fatty metamorphosis of the liver; at least 8 to 10 per cent had marked steatosis prior to performance of the jejunoileal shunt. In preoperative patients, marked fatty metamorphosis was seen most prevalently in the heaviest patients, while the incidence of moderate to minimal fatty infiltration was highest in those not as massively obese. Nevertheless, marked degrees of fatty metamorphosis were seen in patients just meeting the minimal weight criterion for operation, and minimal steatosis or normal livers were seen in more massive candidates. Subsequent to the bypass operation 95 per cent of patients had a moderate to severe degree of fatty metamorphosis apparent within the first 300 days. Subsequently, the incidence of fatty infiltration rapidly decreased so that by 1,500 to 2,000 days the incidence of severe steatosis had returned to that seen preoperatively. In fact, only those patients having associated problems late in the course showed a severe degree of fatty metamorphosis after 1,000 days. A screen of liver function studies was not found helpful in detecting minimal to moderate degrees of fatty metamorphosis and showed only inconsistent changes in those patients with severe steatosis. These tests were not helpful in detecting a quiescent cirrhosis of the liver. Cirrhosis of the liver was seen in two patients. In one, it occurred late in the course after the liver had completely cleared of fat. The cause was thought to be acute alcoholism, and steatosis cleared when drinking ceased. A second patient had a degree of cirrhosis at the time of the bypass operation. There has been slight progress in the ensuing seven or eight years that may be unrelated to the presence of the shortened intestine. Liver function studies do not indicate activity. Because the severe degrees of fatty infiltration seen in patients having previously normal or minimally infiltrated livers occurred during the time of rapid weight loss, it is presumed that the extensive mobilization of depot stores of fat to fulfill caloric requirements of the patient results in accumulation of triglyceride in the liver. Steatosis may be further aggravated by the fact that protein absorption is reduced and, therefore, the formation of low density lipoproteins by the liver is curtailed. In this series, no patients have died as a result of fatty infiltration and liver failure alone.

Alcoholism

Transcriptomic insights into the coordinated regulation of signaling, apoptosis, immunity, and metabolism during Sinonovacula constricta larval metamorphosis.

Metamorphosis is a critical ontogenetic transition for marine bivalves, marking the shift from planktonic to benthic lifestyles, where successful transformation dictates survival. The razor clam Sinonovacula constricta is economically important; however, low larval metamorphosis rates remain a major bottleneck in seedling production. To elucidate the mechanisms governing this process, we performed a comparative transcriptome analysis of S. constricta larvae at pre- and post-metamorphosis stages using Illumina sequencing. A total of 3701 differentially expressed genes (DEGs) were identified, including 3254 up-regulated and 447 down-regulated genes. Functional annotation of the respective top 20 significantly up-regulated and down-regulated DEGs indicated their potential pivotal roles in signal transduction (e.g., up-regulated: CAV1, CHRNA2; down-regulated: APP, NOTCH1), cellular proliferation and differentiation (e.g., up-regulated: TUBA, EGF1; down-regulated: KIF23, TTC25), transcriptional and epigenetic regulation (e.g., up-regulated: NFIL3; down-regulated: OVO, HMX1), substance transport (e.g., up-regulated: LRP2, LRP1B; down-regulated: SLC51A, Slc33a1), substance metabolism (e.g., up-regulated: CPK3, CYP26A1; down-regulated: RDMT1, ADAC), immunomodulation (e.g., up-regulated: CPN2, CRISP2), and protein homeostasis (e.g., up-regulated: HSP27, NAS-27). Functional enrichment analysis further revealed that DEGs were significantly enriched in pathways related to signal transduction and developmental regulation (e.g., Ras, TNF), cell death and homeostasis (e.g., apoptosis), immune responses (e.g., Toll-like receptor), energy metabolism (e.g., lipid), cardiovascular related (e.g., Fluid shear stress), cell junction and architecture (e.g., Tight junction), and infectious disease (e.g., measles). These results suggest a synergistic interplay between signaling, apoptosis, immunity, and metabolism during S. constricta metamorphosis. This study advances our understanding of marine bivalve metamorphosis and offers candidate genes for further mechanistic studies.

Animals

Microrna Expression in Aurelia aurita Metamorphosis.

INTRODUCTION: In animal taxa and jellyfish, the same genome encodes for the different phenotypes that characterize life stages that follow each other during ontogeny. This situation underscores the existence of profound regulation of genomic information at the epigenetic level. MicroRNAs are fundamental epigenetic regulators. The aim of this study is to evaluate the role of microRNA regulation during jellyfish metamorphosis and to explore the existence of evolutionarily conserved microRNAs. METHODS: Specimens belonging to the 4-metamorphosis stages of A. aurita (polyps, ephyra, young, and adult jellyfish) were bred and collected. The expression of 2,549 miRNAs for each stage was tested using microarray technology. The comparison of microRNA expression for each phase was performed using line plot analysis and Principal Component Analysis of variance (PCA), while the identification of microRNA clusters was performed via volcano plot analysis. RESULTS: A remarkable number of A. aurita miRNAs specifically hybridize with a human miRNA library. Each metamorphosis stage is characterized by a different level of expression of miRNAs: 1) Polyp vs. Ephyra stage: 128 upregulated, 2 downregulated; 2) Ephyra vs. Young stage: 2 upregulated, 135 downregulated; 3) Young vs. Adult stage: 69 upregulated, 6 downregulated. Specific functions inferred from known activities of corresponding miRNAs in higher animals (PubMed database) appear to be coherent with the correlated experimental model. DISCUSSION: Present results reveal that microRNAs with human homologs undergo specific expression changes throughout Aurelia aurita metamorphosis. This observation reinforces the hypothesis of a shared evolutionary origin of certain miRNA families between Cnidaria and Bilateria. The dynamic and stage-specific regulation pattern observed suggests that miRNAs play a key role in orchestrating the complex transitions involved in jellyfish development. These findings point to a broader conservation of epigenetic mechanisms, such as miRNA-mediated gene silencing, which may have emerged early in metazoan evolution and contributed to the regulation of cell differentiation and phenotype modulation. CONCLUSION: The present study highlights the importance of Aurelia aurita as a model for investigating miRNA-driven epigenetic regulation in non-bilaterian animals. The identification of human-homologous miRNAs provides novel insights into the evolutionary stability of the epigenetic machinery and suggests conserved regulatory functions across distant taxa. Although limited by the use of a human-based microarray platform, the data presented here lay a solid foundation for future studies employing sequencing and functional assays to further explore the role of miRNAs in cnidarian development and evolution.

Animals

Changes in the tail of Ambystoma maculatum at different stages of metamorphosis: observations on tissue remodeling and its relationship to hydrolytic enzymes.

A system for staging A. maculatum during growth and metamorphosis was devised, based on several parameters of body size; body length, tail length and tail width. Animals at various stages of metamorphosis were employed to study the relationship between specific biochemical and histological changes that occur in the tail of this urodele during metamorphosis. The specific and total activity of two hydrolytic enzymes, acid phosphatase and beta-N-acetyl-glucosaminidase, were measured in tail tissues at progressive stages of development. The activities of these enzymes increased in both the fins and muscular portion of the tail during metamorphosis. These activities can be correlated with resorption of the tail fins and the remodeling of tissues in the muscular portion of the tail.

Acetylglucosaminidase

The generation and changing retinal distribution of displaced amacrine cells in Bufo marinus from metamorphosis to adult.

The generation and retinal distribution of displaced amacrine cells (DAs) were studied from metamorphosis to adult in the cane toad Bufo marinus. Displaced amacrine cells were identified by inducing chromatolytic changes in ganglion cells (GCs) following optic nerve section. Cells that did not chromatolyse in the ganglion cell layer (GCL) of the retina were regarded as DAs. The number of DAs increased considerably from an estimated 10,000 at metamorphosis to 211,000 in the adult toad, and was accompanied by a substantial decrease of average cell density. In contrast to the reported 6:1 cell density gradient of all cells of the GCL in adult toad (Nguyen and Straznicky 1989) only a shallow 1.6:1 density gradient of DAs from the visual streak to the dorsal and ventral retinal margins was detected. Consequently, the incidence of DAs increased from 15% of all cells of the GCL in the visual streak to 30% in the dorsal and ventral peripheral retina. These results indicate that the ratio of the newly generated DAs and GCs at the ciliary margin must be changing during development. More GCs are generated before and around metamorphosis then DAs, in contrast to the relative increase of the percentage of DAs generated after metamorphosis. The possible control of the numbers of DAs in the GCL is discussed.

Animals

Influence of D-penicillamine and beta-aminopropionitril on the metamorphosis of Rana temporaria.

Tadpoles of Rana temporaria (size 5-7 mm) were kept in solutions of D-penicillamine (DPA), beta-aminopropionitril (betaAPN), and L-thyroxine at different concentrations for 18 days. The influence of the substances on metamorphosis was investigated. In low concentrations (1 or 10 mg/100 ml) D-penicillamine accelerates the process of metamorphosis. At these concentrations investigated skeletal malformations could not be observed. A content of 100 mg of DPA/100 ml is toxic. After 8 days of exposure only 4 of the 15 test animals had survived. In all concentrations investigated, beta-aminopropionitril has a toxic action. In low concentration (1 mg/100 ml) deformities are found which could be described as bending of the femur and twisting of the whole hind limb. The animals are non-viable, they die shortly after termination of metamorphosis. Higher concentrations (10 or 100 mg/100 ml) are lethal within the first 10 days. L-thyroxine (0.01 mg/100 ml) shows the wellknown metamorphosis-accelerating effect.

Aminopropionitrile

Golgi studies on Purkinje cell development in the frog during spontaneous metamorphosis. I. General pattern of development.

The development of Purkinje cells was studied in the bullfrog from prometamorphic tadpoles to 10-week-old postmetamorphic froglets by the Golgi-Kopsch method. In this species, the rate of Purkinje cell development is unusually slow and proceeds in two waves. The first wave of development begins prior to the establishment of the external granular layer (EGL), and proceeds slowly for two to three months during the formation of the EGL; then accelerating as metamorphosis is being completed, the cells reach near-adult dimensions a month later. Even prior to the formation of the EGL these cells are already present in the stage of dendritic orientation and flattening which, however, varies from the norm. The second wave of Purkinje cell development begins during metamorphosis and proceeds at a more rapid pace until two months after metamorphosis, at which time they appear to have reached adult dimensions. In these cells the development of the apical dendrite does not always coincide with the stellate stage but may proceed directly to the stage of dendritic orientation and flattening which, in accordance with the norm, is towards the pia and in the sagittal plane. Many variations are present in the dendritic trees and orientation of the dendritic branches of Purkinje cells throughout their development. These variations are similar to those seen in mammals, however, since the frog cerebellum consists of a simple plate, they cannot be attributed to a Cartesian transformation of dendrites to accomodate the curvatures of a folial pattern. Similarly, since these morphological variations occur in the course of normal development they cannot be attributed to a reaction to, or recovery from, injury during development.

Animals

Remodelling of optic nerve myelin sheaths and axons during metamorphosis in Xenopus laevis.

Whole mounts and transverse sections of Xenopus optic nerves were examined with the light and electron microscopes before, during, and after metamorphosis. In stage 52--58 tadpoles, almost all myelin sheaths were circular in transverse sections. Early in metamorphosis (stages 60--61) large redundant myelin loops surrounded many large axons in central regions of the nerve. The loops subsequently were broken down into ovoids and lamellar segments that remained mostly within oligodendrocytes. These myelin changes were not observed in the chiasm or next to the eye. They were not associated with significant axonal degeneration and were no longer apparent in optic nerves of young frogs. Xenopus optic nerves also became shorter during metamorphosis. We therefore suggest that myelin sheaths with redundant loops which degenerate and disappear are being remodelled as the nerve decreases in length.

Animals

Daily variations in the effects of disturbance on growth, fattening, and metamorphosis in the bullfrog (Rana catesbeiana) tadpole.

Bullfrog tadpoles subjected to the daily disturbance of water replacement demonstrated differences in the rates of growth and metamorphosis and in fatbody weights which depended on the time of day when water replacement occurred. Early daily disturbances stimulated larval growth whereas midday disturbances stimulated metamorphosis. The time when the daily disturbances caused fattening changed during metamorphosis so that younger tadpoles fattened when disturbed early in the photoperiod and older tadpoles fattened when disturbed at midday.

Adipose Tissue

Monoamines and their metabolites in the amphibian (Ambystoma tigrinum) brain: quantitative changes during metamorphosis and captivity.

1. Monoamine neurotransmitters (epinephrine, norepinephrine, dopamine, serotonin and some of their metabolites (DOPEG, MHPG, DOPAC, 5-HIAA) were measured by HPLC in extracts from telencephalon (TEL) and diencephalon-midbrain (DM) before, during at the end of metamorphosis. 2. During metamorphosis MHPG increased and 5-HIAA decreased in TEL and DM while DOPEG decreased only in DM. 3. Monoamine levels were greater in the TEL and a larger increase in MHPG occurred there. 4. Captivity without metamorphosis also caused a significant depression of 5-HIAA in TEL and depression of DOPEG, MHPG and DOPAC in DM.

Ambystoma

DNA synthesis and turnover in the bullfrog tadpole during metamorphosis.

125I-labeled deoxyuridine (IdUrd) has been used to estimate the turnover of DNA in liver, tail, and hind limb during spontaneous and triiodothyronine-induced metamorphosis. It was found that the total amount of liver DNA remained constant and there was no significant loss of the label from the liver DNA, which would be expected if there was an increase in DNA turnover during metamorphosis. Also, the change in specific activity of liver DNA parallels that of tail DNA during spontaneous metamorphosis. These data suggest that metamorphic transitions in the tadpole liver do not involve significant changes in DNA turnover. It was observed that the incorporation of label into hind limb DNA showed a high variability among individual animals as compared to liver and tail tissue. The data presented suggest that the observed variability is not a random phenomenon but related directly to the rate at which animals will metamorphose.

Animals

Fiber counts of regenerating peripheral nerves in axolotls and the effect of metamorphosis.

Counts have been made of myelinated and unmyelinated fibers in the sixteenth nerve root of the axolotl, before, during and after regeneration following nerve section. There is no loss of myelinated fibers. Many collateral sprouts are formed and eventually withdrawn. Regeneration during metamorphosis is similar but there is more sprouting. There is no fiber loss from cut nerves in metamorphosis. This suggests that rising thyroid hormone levels are not in themselves detrimental to nerves without peripheral connections. Other factors that may govern cell loss in limb development are discussed.

Ambystoma

Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.

Amino Acid Sequence