Late Prometamorphosis Research Articles

Developmental expression profiles and thyroidal regulation of cytokines during metamorphosis in the amphibian Xenopus laevis

Early life-stages of amphibians rely on the innate immune system for defense against pathogens. While thyroid hormones (TH) are critical for metamorphosis and later development of the adaptive immune system, the role of TH in innate immune system development is less clear. An integral part of the innate immune response are pro-inflammatory cytokines – effector molecules that allow communication between components of the immune system. The objective of this study was to characterize the expression of key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and interferon-γ (IFN-γ), throughout amphibian development and determine the impacts of thyroidal modulation on their expression. Xenopus laevis were sampled at various stages of development encompassing early embryogenesis to late prometamorphosis and cytokine expression was measured by real-time PCR. Expression of TNFα and IL-1β were transient over development, increasing with developmental stage, while IFN-γ remained relatively stable. Functionally athyroid, premetamorphic tadpoles were exposed to thyroxine (0.5 and 2 μg/L) or sodium perchlorate (125 and 500 μg/L) for seven days. Tadpoles demonstrated characteristic responses of advanced development with thyroxine exposure and delayed development (although to a lesser extent) and increased thyroid gland area and follicular cell height with sodium perchlorate exposure. Exposure to thyroxine for two days resulted in decreased expression of IL-1β in tadpole trunks. Sodium perchlorate had negligible effects on cytokine expression. Overall, these results demonstrate that cytokine transcript levels vary with stage of tadpole development but that their ontogenic regulation is not likely exclusively influenced by thyroid status. Understanding the direct and indirect effects of altered hormone status may provide insight into potential mechanisms of altered immune function during amphibian development.

Analysis of thyroid hormone receptor βA mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors alpha (TRalpha) and betaA (TRbetaA) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TRalpha gene expression whereas a marked up-regulation of TRbetaA mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TRbetaA mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TRbetaA mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TRbetaA gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TRbetaA expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TRbetaA mRNA up-regulation. Results of this study suggest that TRbetaA mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.

Regression of blood vessels in the ventral velum of Xenopus laevis Daudin during metamorphosis: light microscopic and transmission electron microscopic study.

Structural changes of the ventral velum of Xenopus laevis tadpoles from late prometamorphosis (stage 58) to the height of metamorphic climax (stage 62) were examined by light and transmission electron microscopy. Special emphasis was given to the blood vessel regression. Early changes of velar capillaries were formation of luminal and abluminal endothelial cell processes, vacuolation, and cytoplasmic and nuclear chromatin condensation. At the height of metamorphic climax, transmission electron microscopy revealed apoptotic endothelial cells with nuclear condensation and fragmentation, intraluminal bulging of rounded endothelial cells which narrowed or even plugged the capillary, and different stages of endothelial cell detachment ('shedding') into the vessel lumen. These changes explain the 'miniaturisation' of the velar microvascular bed as well as the typical features found in resin-casts of regressing velar vessels which have been observed in a previous scanning electron microscopy study of the ventral velum.

Metamorphosis in the summer flounder, Paralichthys dentatus: thyroidal status influences salinity tolerance.

Metamorphosis in the summer flounder (Paralichthys dentatus) is controlled by thyroid hormones (TH) and takes place as the larvae move from a salinity of about 35 parts per thousand (ppt) in the ocean to salinity ranging from 0-35 ppt in estuaries. Historically, the role of TH in juvenile and adult teleost osmoregulation has been ambiguous, and it is not known if TH influences larval teleost osmoregulatory development. This study addresses the influence of thyroxine (T4) on the development of tolerance to low (5 ppt) and high salinity (45 and 50 ppt) as determined by salinity tolerance tests. In untreated larvae, tolerance to both low and high salinity was high during early premetamorphosis (early pre-M) and decreased or was very low from late prometamorphosis (late pro-M) through mid-metamorphic climax (mid-MC). Salinity tolerance increased 2-3-fold during late MC when whole-animal T4 levels are highest, and reached maximum tolerance at the juvenile stage. The early induction of metamorphosis by exposing larvae in pre-M to exogenous T4 reduced tolerance to low salinity during early and mid-MC, though tolerance of fish that had developed into juveniles was not impaired. In contrast, T4 increased high salinity tolerance during early and mid-MC, and the juvenile stage. This T4-induced heterochrony in salinity tolerance with regards to developmental stage suggests that the effects of T4 on salinity tolerance may be uncoupled from accelerated metamorphosis. Treatment of larvae with thiourea (TU, an inhibitor of T4 synthesis) inhibited metamorphosis and reduced tolerance to high salinity, but did not affect tolerance to low salinity. Reduced tolerance to high salinity by TU was only partially counteracted by T4 treatment, suggesting that TU also affects hypoosmoregulatory activity by an extrathyroidal mechanism. Our findings suggest that in the summer flounder T4 plays a more important role in the development of hypoosmoregulatory ability than hyperosmoregulatory ability. J. Exp. Zool. 284:414-424, 1999.

Acceleration of Anuran Amphibian Metamorphosis by Corticotropin-Releasing Hormone-like Peptides

Despite substantial information on the role of the pituitary-thyroid and pituitary-interrenal axes in controlling amphibian metamorphosis, the hypothalamic hormones responsible for controlling the activity of these axes have not been identified. The mammalian thyrotropin-releasing hormone (TRH) does not regulate the thyroid axis of tadpoles; however, corticotropin releasing hormone (CRH) stimulates the release of thyrotropin from bullfrog tadpole pituitary glands in vitro and may thus function as a central regulator of the thyroid axis during metamorphosis. I tested the possibility that a CRH-like peptide is involved in controlling amphibian development by treating tadpoles of two anuran species, the western spadefoot toad Scaphiopus hammondii , and the North American bullfrog, Rana catesbeiana, with neuropeptides and monitoring their effects on metamorphosis. Injection of spadefoot toad tadpoles with ovine (o) CRH (2 μg/animal every other day for 3 weeks) or the amphibian CRH-like peptide sauvagine (SV) significantly decreased their time from hatching to metamorphic climax (Gosner stage 42; frontlimb emergence) and their body weight and body length at climax compared with vehicle-injected controls: whereas, TRH had no effect and arginine vasotocin produced a small but significant lengthening of the larval period but did not alter body size at climax. In an acute response experiment, S. hammondii tadpoles (in Gosner stages 36-38—late prometamorphosis) treated with oCRH or SV (2 μg/animal) exhibited significantly elevated whole-body thyroxine (T 4) content at 2 and 6 hr after injection; whereas, treatment with TRH (2 μg/animal) did not significantly alter whole-body T 4. R. catesbeiana tadpoles treated with oCRH or SV (surgical implantation of ELVAX pellets impregnated with 100 μg peptide and injections of peptides at 5 μg/animal once every 3 days) exhibited accelerated spontaneous and triiodothyronine (T 3)induced metamorphosis as assessed by changes in tail height, hind limb development, and body weight; TRH had no effect. Injections of a pool of antisera generated against CRH-like peptides (rat/human CRH. oCRH, SV) slowed T 3-induced metamorphosis when compared with normal serum-injected controls. These results support the hypothesis that a CRH-like peptide(s) is involved in the central control of metamorphosis of anuran amphibians, and may act, at least in part, through stimulation of the thyroid axis.

Organization of tyrosine hydroxylase-immunoreactive neurons in the di- and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis.

We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di- and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.

Ontogeny of immunoreactive gonadotropin-releasing hormone neuronal systems in amphibians

The ontogeny of gonadotropin-releasing hormone (GnRH) systems was investigated in 3 anuran amphibians (genus Rana) by means of immunocytochemical (ICC) techniques and antibodies generated against 3 different forms of GnRH. Antisera that recognize primarily chicken II and mammalian GnRHs revealed two anatomically and developmentally distinct GnRH systems. One system, referred to here as the forebrain-spinal cord system, contained GnRH immunoreactive (ir) fibers extending from the rostral diencephalon through the ventromedial brainstem to the spinal cord. Intensity of labeling was robust in the youngest, premetamorphic tadpoles, but decreased with age. GnRH immunolabeling in the hypothalamic-pituitary tract was not detected until late prometamorphosis and increased with age. Development of GnRHir in the hypothalamic-pituitary tract coincided with first appearance of GnRHir in the terminal nerve in R. catesbeiana, but not in R. cascadae or R. aurora, suggesting species differences. Comparisons of results obtained with antisera to different forms of GnRH support the interpretation that the forebrain-spinal cord system, hitherto undescribed in amphibians, develops first and synthesizes a non-mammalian, chicken II-like GnRH, and that the hypothalamic-pituitary system develops later and synthesizes primarily mammalian GnRH. We speculate that the forebrain-spinal cord system may represent a GnRH innervation of frog sympathetic ganglia, and that the two GnRH systems are chemically and embryonically distinct.

Immunohistochemical localization of corticotropin-releasing factor- and arginine vasotocin-like immunoreactivities in the brain and pituitary of the American bullfrog ( Rana catesbeiana) during development and metamorphosis

Immunoperoxidase staining for corticotropin-releasing factor (CRF) in the median eminence was sparce or absent from premetamorphic tadpoles, but increased dramatically by late prometamorphosis. Quantitative photometry revealed that CRF-like immunostaining material in the median eminence was most dense in metamorphic climax tadpoles. Arginine vasotocin (AVT)-like immunostaining material was visualized in perikarya of the magnocellular nucleus, with extensive fiber staining seen in the medial basal and infundibular hypothalamus as well as in the median eminence and pars nervosa of the pituitary. AVT-like immunoreactive perikarya were virtually absent in premetamorphic tadpoles, but their number increased greatly by Taylor-Kollros stage XII and continued to increase after this stage. Quantitative photometry revealed that AVT-like immunoreactivity in the pars nervosa increased greatly at Taylor-Kollros stage XII and remained intense after this stage. AVT-like immunoreactivity did not appear in the median eminence until Taylor-Kollros stage XVI. Localization of AVT-like immunoperoxidase staining around portal vessels in the median eminence suggests an anatomical mechanism for delivery of AVT to anterior pituitary corticotropes. These results indicate that both CRF and vasotocinergic neuronal systems develop just before the activation of interrenal steroidogenesis which occurs during the later stages of metamorphosis in this species.

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis.

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.

Putative nuclear triiodothyronine receptors in tadpole erythrocytes during metamorphic climax.

The characteristics of the nuclear T3 receptors present in red blood cells (RBCs) of Rana catesbeiana tadpoles undergoing metamorphic climax have been investigated with a T3 saturation technique. Because there were significant amounts of receptor-bound endogenous hormone, prolonged incubation in vitro (48 h at 21 C) was necessary to achieve binding equilibrium. Receptor number, which averaged 783 +/- 35 (+/- SE) sites/nucleus during early prometamorphosis (stages XIV-XVI), increased rapidly during the subsequent stages of this phase. By stage XIX, receptor number had reached a maximum of 2464 +/- 152. During climax, receptor number decreased steadily, and by stage XXV, it was lower than that observed during premetamorphosis (stages V-X). This decrease was attributed to the replacement of larval RBCs, which have a high receptor content, by adult RBCs, which possess significantly fewer sites per nucleus. At midclimax, adult and larval RBCs were separated on a Renografin continuous density gradient. Adult cells constituted more than 65% of the total cell population and were shown to contain only 126 +/- 46 sites/nucleus. Although the striking increase in receptor number preceded the substantial increases in plasma T4 and T3 levels that occurred during climax, it was associated with a measurable increase in plasma thyroid hormone levels. Furthermore, receptor number in stage XIV tadpoles was increased markedly after immersion for 14 days in water containing sufficient T3 to raise plasma T3 only to levels normally observed in late prometamorphosis. These findings strongly support the hypothesis that the increases in receptor number and plasma thyroid hormone levels that occur before climax in this species are causally related.

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 3 H-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.

Lipid changes in the tadpoles of Rana tigrina during growth and metamorphosis

1. 1. Total lipids, neutral lipids and phospholipids of tadpoles of Rana tigrina were studied during their growth and metamorphosis. 2. 2. Total lipids increased during the growth period and early prometamorphosis but decreased during late prometamorphosis and metamorphosis. In young adult growth stages they showed a gradual increase. 3. 3. Neutral lipids consisted of triglycerides as the major fraction, cholesterol, cholesterol esters and diglycerides in moderate concentration and monoglycerides and free fatty acids in low concentration. Quantitative alterations in neutral lipids and all the components except fatty acids paralleled those occurring in total lipids. Fatty acids increased during the growth period and entire prometamorphosis and early metamorphosis but showed some decrease in late metamorphosis. 4. 4. Phosphatidyl choline and phosphatidyl ethanolamine were major phospholipid components. Phosphatidyl inositol, phosphatidyl serine and spingomyelin occurred in moderate concentrations, whereas lysophosphatidyl choline and phosphatidic acid occurred in trace amounts. Quantitative alterations in phospholipids and their individual components paralleled those observed in total and neutral lipids. 5. 5. Composition and alterations in total, neutral and phospholipids of anuran tadpoles in growth and metamorphosis are compared with the same in the insect larvae in growth and metamorphosis. 6. 6. The significance of alterations in lipids during histogenesis and histolysis occurring in anuran metamorphosis is presented and discussed.

β-Glucoronidase in the excreta of tadpoles of Rana tigrina during growth and metamorphosis

1. 1. β-Glucuronidase activity in the excreta of tadpoles was very low in growth and premetamorphosis, but during late prometamorphosis it showed a sixteenfold increase, whereas during metamorphosis it increased by ninetyfold. In young adults there was a considerable decrease. 2. 2. β-Glucuronidase was specifically localized in the lysosomes in the degenerated intestinal mucosa cells found in the excreta. 3. 3. β-Glucuronidase showed single pH optimum at 4·2, a single decay in thermal inactivation indicating its heat-stable nature and different responses to activators and inhibitors. 4. 4. Properties of β-glucuronidase in excreta are compared with those of β-glucuronidase found in regressing tail, liver and degeneration of intestinal mucosa of tadpoles during metamorphosis, and with those of β-glucuronidase found in insect metamorphosis. 5. 5. The behaviour of β-glucuronidase in excreta is compared with that in the metamorphic reshaping of the alimentary canal. Mechanisms for getting rid of enhanced lysosomal enzymes after metamorphosis are discussed comparatively in anurans and insects.

β-Glucuronidase in alimentary canal of tadpoles of Rana tigrina during growth and metamorphosis

1.1. β-Glucuronidase activity of the alimentary canal of tadpoles showed an eightfold increase during early prometamorphosis and a significant decrease in late prometamorphosis. During early metamorphosis it again showed a fivefold increase and in late metamorphosis a twofold decrease.2.2. In early prometamorphosis β-glucuronidase was localized in phagosomes in degenerating epithelium and resorbing anal canal piece but in metamorphosis it showed diffused cytoplasmic localization.3.3. Significant differences in pH optima and response to activators and inhibitors by β-glucuronidase in early prometamorphosis and metamorphosis were observed.4.4. β-Glucuronidase plays a lytic role in degeneration of intestinal mucosa and resorption of anal canal piece but in reconstitution of mucosa it has a different role.

Hypothalamic neurosecretion and metamorphosis in Xenopus laevis. I. The effect of propylthiouracil.

Normal and propylthiouracil (PTU) treated Xenopus laevis tadpoles were fixed during all stages of metamorphosis and sagittal sections were stained with aldehyde fuchsin (AF) or pseudoisocyanine (PIC). Whereas AF positive neurosecretory material could only be demonstrated in the preoptic nucleus from late prometamorphosis, increasing amounts of PIC positive material were found in cells of the dorsal part of the preoptic region from early premetamorphosis. The development of these cells correlated with that of the thyrotropic cells and the thyroids. Likewise, signs of hyperactivity in thyroids and thyrotropic cells of PTU treated larvae were accompanied by a depletion of the dorsal PIC positive cells. In the ventral preoptic region PIC positive cells developed from late prometamorphosis in control larvae, but failed to do so in PTU treated animals. It is argued that the differentiation of the PIC positive cells is largely dependent on thyroid hormones; that the dorsal PIC positive cells may produce a thyrotropin releasing factor; and that the function of these dorsal cells is inhibited by thyroid hormones.