Variation in thyroid hormone action and tissue content underlies species differences in the timing of metamorphosis in desert frogs

Hormonal control of post-embryonic morphogenesis is well established, but it is not clear how differences in developmental endocrinology between species may underlie animal diversity. We studied this issue by comparing metamorphic thyroid hormone (TH) physiology and gonad development across spadefoot toad species divergent in metamorphic rate. Tissue TH content, in vitro tail tip sensitivity to TH, and rates of TH-induced tail tip shrinkage correlated with species differences in larval period duration. Gonad differentiation occurred before metamorphosis in species with long larval periods and after metamorphosis in the species with short larval periods. These differences in TH physiology and gonad development, informed by phylogeny and ecology of spadefoot metamorphosis, provide evidence that selection for the short larval periods in spadefoot toads acted via TH physiology and led to dramatic heterochronic shifts in metamorphic climax relative to gonad development.     

Histology and microscopic anatomy of the thyroid gland during the larval development of Pseudis platensis (Anura,Hylidae)

Several hormones regulate anuran larval development, most notably thyroid hormones (THs). In anurans, metamorphosis fails when the thyroid gland is absent or inactivated, resulting in giant tadpoles. Larval gigantism occurs naturally in neotropical frogs of the genus Pseudis as a result of a prolonged larval period. Its thyroid function is poorly investigated and the focus of this study. We describe qualitative and quantitative variations in larval development for field-captured specimens of Pseudis platensis and compare those to the development of two sympatric species, Phyllomedusa sauvagii and Pithecopus azureus, which have small tadpoles and a shorter larval period. We describe morphological changes in the thyroid glands of larval and adult specimens. In contrast to other species with similar ecological requirements, P. platensis exhibits distinct glandular activity. During premetamorphosis, there was little or no thyroid activity, a period in which the tadpole reached 70% of its maximum size. Development and degree of activity of the thyroid gland determine the duration of the early stages of the larval period. Thyroid gland histology in tadpoles appears to correlate with the TH activity, and in turn with the diversity in anuran life history transitions.     

Histo-morphology of the thyroid gland during the larval development of Pleurodema borellii (Anura,Leptodactylidae)

The thyroid gland is essential in anurans, since thyroid hormones (TH) are the main regulators of larval development. Its absence or inactivity interrupts development and precludes metamorphosis. Histological changes are important diagnostic criteria for evaluating thyroid gland activity. However, there is a large larval diversity where the development of the thyroid gland development has not been studied. Pleurodema borellii is an anuran from northwest of Argentina with typical omnivorous pond tadpoles that can be easily raised in captivity. This study explores the histo-morphological changes of the thyroid gland architecture during larval development. Histological parameters indicate peak glandular activity in parallel with the intensity of the metamorphic transformations. These parameters regress towards the end of metamorphosis, indicating low TH release. P. borellii's thyroid gland does not appear to have relevant activity in post-metamorphic juvenile stages. This study is a first step towards understanding endocrine regulation during the development of Pleurodema borellii, and a reference to future studies in this species involving thyroid-dependent processes.     

Microvascularization of the thyroid glands in larval and adult Xenopus laevis‐ histomorphology and scanning electron microscopy of vascular corrosion casts

This study demonstrates gross arterial supply, venous drainage and microvascular patterns of larval and adult thyroid glands in the African Clawed Frog, Xenopus laevis by scanning electron microscopy of vascular corrosion casts and light microscopy of stained serial tissue sections. Results confirm published findings gained by microscopical dissections with respect to gross arterial supply. However, in adult frogs one rather than two thyroid veins are found. This study reveals for the first time that bilaterally located thyroid glands in premetamorphosis have immature capillary networks, lack a clear hierarchy of blood vessels, and show many signs of sprouting angiogenesis. During metamorphic climax, blood vessels gain a clear hierarchy and capillaries form closed networks around thyroid follicles. From climax onwards, non‐sprouting angiogenesis (intussusceptive microvascular growth) becomes the prevailing mode of angiogenesis intensifying follicle capillarization. Due to narrow interfollicular spaces, thyroidal arterioles remain superficial while draining venules are located interfollicularly. In contrast with the mammalian thyroid gland where most thyroid follicles have their own capillary beds, most thyroid follicles in Xenopus share their capillary beds with neighbouring follicles. Consequently, the concept of individual morphological and functional angiofollicular units applicable to adult mammalian and human thyroid glands is not applicable to larval and adult amphibian thyroid glands.     

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。       

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-Lthyronine have profound effects on postenbryonic development of most vertebrates.Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action.Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis,cell death,re-structuring,etc.) in free-living embryos and larvas of most anurans.This article will first describe the salient features of metamorphosis and its control by TH and other hormones.Emphasis will be laid on the key role played by TH receptor (TR),in particular the phenomenon of TR gene autoinduction,in initiating the developmental action of TH.Finally,it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

Intraspecific developmental variation in the life cycle of the Andean Treefrog (Boana riojana): A temporal analysis

Intraspecific variation during the anuran larval period has been analyzed mainly in relation to the timing of metamorphosis and body size at metamorphosis. However, other traits may vary as well. We examined two developmental series of Boana riojana from the same population in two consecutive years and describe intraspecific variation in larvae of this species. We discuss how variation, if present, may influence its life cycle. We found that both larval series differed in the larval period length, one twice as long as the other. This variation primarily depended on when breeding occurred, metamorphosis was achieved during late spring in both generations and at similar sizes, and only the rate of larval development during premetamorphosis varied extensively between years. This is consistent with thyroid gland activity because when it became active the developmental trajectory became more canalized. No variation of staging sequence occurred in relation to the different durations of the larval period. However, in the long-lasting series we found two different morphs. Also, integument, thyroid gland, skeleton, and testis differentiation events occurred at the same developing stages. In contrast, ovarian differentiation proceeded at the same absolute age in both series. Sexual dimorphism becomes evident within the year after metamorphosis. The intraspecific heterochrony that we describe for the larval development of B. riojana does not lead to phenotypic variation at the end of metamorphosis. We discuss the importance of analyzing growth and development independently. Each proceeds differently in time, but with an interdependence at some point, because size and shape do not vary at the end of metamorphosis.     

Regulation of Metamorphosis-Associated Changes in the Lipid Metabolism of Selected Vertebrates

SYNOPSIS. The life histories of many vertebrates include complex,postembryonic developmental pathways that involve morphologicaland physiological changes that adapt juveniles to a new habitat.A survey of such developmental pathways, including lamprey metamorphosis,salmonid smoltification, and anuran metamorphosis, reveals acommon strategy of lipid metabolism consisting of two distinctphases. The first phase is characterized by lipid accumulationin storage sites and resultsfrom lipogenesis prevailing overlipolysis. The second phase is characterized by lipid depletionfrom storage sites and results from lipolysis prevailing overlipogenesis. Regulation of lipid deposition and lipid mobilizationis essential for ensuring availability of lipid during timesof need. Lipogenesis is promoted by insulin and, in lampreyand anurans, also by thyroid hormones. Lipolysis is promotedby a number of hormones, including prolactin, growth hormone,adrenocorticotropic hormone, corticosteroids, somatostatins,and thyroid hormones. The coordinate regulation of development-associatedchanges in lipid metabolism results from interactions amonghormones and other internal and environmental cues.     

Growth and metamorphosis of the rectus abdominis muscle in Rana pipiens

Cross sections through the middle segment of the anuran rectus abdominis muscle were analyzed morphometrically at nine stages of development, from early larval life through full maturity. The numbers, sizes, and relative distributions of twitch and slow muscle fibers, newly differentiated fibers, degenerating fibers, and satellite cells were determined at each stage. The data indicate that the muscle increases slowly in size and fiber content during early larval life. New fibers appear to form primarily along the medial margin of the muscle. During mid-larval stages, when thyroid hormone levels are rising, new fibers form throughout the medial portion of the muscle. At a slightly later stage, fibers in the lateral region of the muscle begin to degenerate. Structurally normal presynaptic elements are present on both degenerating fibers and the empty basal laminae of fibers that had been removed by phagocytes. Both fiber formation and fiber loss slow during midmetamorphic climax, at the time when thyroid hormone levels reach a peak in anurans and begin to decline. Degenerating fibers appear within the body of the muscle at the end of metamorphosis. By the end of the second postmetamorphic month, neither degenerating nor newly differentiated fibers are present. The muscle continues to grow through adult life primarily by fiber hypertrophy.     

Effect of changes in resource level on age and size at metamorphosis in Hyla squirella

Recent experiments suggest that timing of metamorphosis is fixed during development in some anurans, insects, and freshwater invertebrates. Yet, these experiments do not exclude a growth rate optimization model for the timing of metamorphosis. I manipulated food resources available to larvae of squirrel treefrogs (Hyla squirella) to determine if there is a loss of plasticity in duration of larval period during development and to critically test growth rate models for the timing of metamorphosis. Size-specific resource levels for individual tadpoles were switched from low to high or high to low at three developmental stages spaced throughout larval development. The effects of changes in resource availability on larval period and mass at metamorphosis were measured. Switching food levels after late limb bud development did not significantly affect larval period in comparison to constant food level treatments. Therefore, developmental rate in H. squirella is better described by a fixed developmental rate model, rather than a growth rate optimization model. The timing of fixation of developmental rate in H. squirella is similar to that found in other anuran species, suggesting a taxonomically widespread developmental constraint on the plasticity of larval period duration. Mass at metamorphosis was not significantly affected by the timing of changes in food levels; the amount of food available later in development determined the size at metamorphosis. Larval period and mass at metamorphosis were negatively correlated in only one of two experiments, which contrasts with the common assumption of a phenotypic trade-off between decreased larval period and increased mass at metamorphosis. Received: 19 August 1996 / Accepted: 20 June 1997     

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

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

Altered thyroid hormone levels affect the capacity for temperature-induced developmental plasticity in larvae of Rana temporaria and Xenopus laevis

Anuran larvae show phenotypic plasticity in age and size at metamorphosis as a response to temperature variation. The capacity for temperature-induced developmental plasticity is determined by the thermal adaptation of a population. Multiple factors such as physiological responses to changing environmental conditions, however, might influence this capacity as well. In anuran larvae, thyroid hormone (TH) levels control growth and developmental rate and changes in TH status are a well-known stress response to sub-optimal environmental conditions. We investigated how chemically altered TH levels affect the capacity to exhibit temperature-induced developmental plasticity in larvae of the African clawed frog (Xenopus laevis) and the common frog (Rana temporaria). In both species, TH level influenced growth and developmental rate and modified the capacity for temperature-induced developmental plasticity. High TH levels reduced thermal sensitivity of metamorphic traits up to 57% (R. temporaria) and 36% (X. laevis). Rates of growth and development were more plastic in response to temperature in X. laevis (+30%) than in R. temporaria (+6%). Plasticity in rates of growth and development is beneficial to larvae in heterogeneous habitats as it allows a more rapid transition into the juvenile stage where rates of mortality are lower. Therefore, environmental stressors that increase endogenous TH levels and reduce temperature-dependent plasticity may increase risks and the vulnerability of anuran larvae. As TH status also influences metabolism, future studies should investigate whether reductions in physiological plasticity also increases the vulnerability of tadpoles to global change.     

Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus

MiR-1, miR-133a, and miR-206a have been identified as muscle-specific miRNAs. They play multiple crucial roles in the regulation of muscle development. Here, we show that these miRNAs were differentially expressed during the larval development of flounder, and specifically expressed in skeletal muscle and heart in adult tissues/organs. The expression levels of these miRNAs were significantly changed by thyroid hormone (TH) or thiourea (TU) treatment during metamorphosis from 17 dph (days post hatching) to 42 dph. In addition, the expression levels of MyoD and Myf5 mRNAs markedly increased at 14 dph (pre-metamorphosis) compared to metamorphic stages, and their expression levels are far above the myogenin during larval development. Moreover, these MRFs (myogenic regulatory factors) expression were directly or indirectly regulated by thyroid hormone or thiourea during metamorphosis. All the results suggest that miRNAs and MRFs might be involved in signaling pathway of TH or TU-mediated flounder metamorphosis.     

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP-9TH, similar to the previously reported Xenopus laevis MMP-9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP-9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone-treated tail-derived myoblastic cultured cells, while MMP-9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP-9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP-9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone.

The thyroid hormones L-thyroxine and triiodo-L-thyronine have profound effects on postembryonic development of most vertebrates. Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process (morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. This article will first describe the salient features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the key role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis

Abstract. Metamorphosis in the South African clawed frog, Xenopus laevis , is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T₄) and triiodothyronine (T₃). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH.
These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH.
Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.     

The morphology of amphibian skin vascularization before and after metamorphosis

Summary The skin vascularization was investigated troughout the ontogenetic development and in adults of two anurans, Rana temporaria and Bufo bufo, and two urodeles, Triturus vulgaris and Triturus cristatus. It was found that, contrary to the urodele larvae, the anuran tadpoles have a very sparse skin vascularization. During the early stages of anuran metamorphosis the skin capillary network becomes dense; later, during skin metamorphosis, a second, venous, network is formed as anastomoses between the subcutaneous vein ramifications. In the urodeles, metamorphosis is not accompanied by any significant morphological changes in the skin vascularization, and a subcutaneous network is not formed. It is suggested that the reduced skin vascularization in anuran tadpoles is an advanced larval character relative to the rich vascularization of the skin in urodele larvae. It is further suggested that anuran tadpoles have a reduced ability to utilize gaseous exchange through the skin. The function of the subcutaneous venous network found in anurans after metamorphosis is obscure; experiments indicate a vasomotor regulation which is neither adrenergic nor cholinergic.Abbreviations a arteriole - A artery - an anastomosis between the subepidermal capillary network and the subcutaneous venous network - C stratum compactum - E epidermis - ec subepidermal capillaries - S stratum spongiosum - sv subcutaneous venous network - v venule - V vein - vc venae comitantes     

Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae

Tadpoles that spontaneously arrest development and remain as larvae occur occasionally in Xenopus laevis populations. These non-metamorphosing tadpoles continue to grow, and they develop into grossly deformed giant individuals which come as close as any anurans to being truly neotenic. Giant X. laevis tadpoles that fail to metamorphose lack thyroid glands. In this study, the hypothesis that the tissues of these tadpoles nevertheless remain thyroid hormone sensitive was tested, by exposing isolated tadpole tail tips to exogenous thyroid hormone in tissue culture. The tail tips from giant tadpoles significantly shrank in response to the thyroid hormone treatment, showing that their tissue was still capable of metamorphosis. However, the amount of shrinkage was less than that observed in tail tissue from normal tadpoles. It was hypothesized that complete induction of metamorphosis may not be possible in the giant tadpoles due to a disproportionate growth and development of tissues and organs.