Cultured cells as a model for amphibian metamorphosis.

Gene expression screens have been applied to a cultured cell line of Xenopus laevis, XL-177, to isolate genes that are up- and down-regulated in the first 8 h after thyroid hormone (TH) induction. At least 14 up-regulated genes were isolated from TH-induced cells grown in the presence or absence of cycloheximide, an inhibitor of protein synthesis. These genes respond directly to TH as demonstrated by the resistance of up-regulation to protein synthesis inhibition in the cultured cells or in tadpoles. Kinetics of mRNA accumulation after TH induction is similar for these genes, including those that are superinduced by cycloheximide. Their mRNAs start to be up-regulated several hours after TH treatment and reach maximum levels between 8 and 16 h. These genes show up-regulation in one or more tadpole organs in response to exogenous TH. Only a few minimally down-regulated genes were identified. Fourteen of the 20 genes that were found to be up-regulated by TH in tadpole tail are also up-regulated in XL-177 cells. Their up-regulation falls into the same two kinetic patterns in the cultured cells as it does in tadpole tail. Another cell line of X. laevis, XLA, is greatly reduced in its ability to up-regulate the same genes isolated from XL-177 cells and tadpole tails in response to TH. Thus these cell lines make up a model system to examine the interactions of gene expression triggered by TH during amphibian metamorphosis.     

Apoptotic and survival signaling mediated through death receptor members during metamorphosis in the African clawed frog Xenopus laevis

The tumor necrosis factor (TNF) superfamily includes death receptor (DR) ligands, such as TNF-α, FasL, and TRAIL. Death receptors (DRs) induce intracellular signaling upon engagement of their cognate DR ligands, either leading to apoptosis, survival, or proinflammatory responses. The DR signaling is mediated by the recruitment of several death domain (DD)-containing molecules such as Fas-associated death domain (FADD) and receptor-interacting protein (RIP) 1. In this review, we describe DR signaling in mammals, and describe recent findings of DR signaling during metamorphosis in the African clawed frog Xenopus laevis. Specifically, we focus on the cell fate (apoptosis or survival) mediated through a DR ligand, TNF-α or TRAIL in endothelial cells or red blood cells (RBCs). In addition, we discuss relationships between thyroid hormone-induced metamorphosis and DR signaling.     

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3'-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T(3) is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit beta gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.     

Amphibian metamorphosis as a model for studying endocrine disruption on vertebrate development: Effect of bisphenol A on thyroid hormone action

Thyroid hormone (TH) is essential for proper development in vertebrates. TH deficiency during gestation and early postnatal development produces severe neurological, skeletal, metabolism and growth abnormalities. It is therefore important to consider environmental chemicals that may interfere with TH signaling. Exposure to environmental contaminants that disrupt TH action may underlie the increasing incidence of human developmental disorders worldwide. One contaminant of concern is the xenoestrogen bisphenol A (BPA), a chemical widely used to manufacture polycarbonate plastics and epoxy resins. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. As TH action at the cellular level is highly conserved across vertebrate species, amphibian metamorphosis serves as an important TH-dependent in vivo vertebrate model for studying potential contributions of BPA toward human developmental disorders. Using Xenopus laevis as a model, we and others have demonstrated the inhibitory effects of BPA exposure on metamorphosis. Genome-wide gene expression analysis revealed that surprisingly, BPA primarily targets the TH-signaling pathway essential for metamorphosis in Xenopus laevis. Given the importance of the genomic effects of TH during metamorphosis and the conservation in its regulation in higher vertebrates, these observations suggest that the effect of BPA in human embryogenesis is through the inhibition of the TH pathway and warrants further investigation. Our findings further argue for the critical need to use in vivo animal models coupled with systematic molecular analysis to determine the developmental effects of endocrine disrupting compounds.     

Corticotropin-releasing factor is cytoprotective in Xenopus tadpole tail: coordination of ligand, receptor, and binding protein in tail muscle cell survival

Upon metamorphosis, amphibian tadpoles lose their tails through programmed cell death induced by thyroid hormone (T3). Before transformation, the tail functions as an essential locomotory organ. The binding protein for the stress neuropeptide corticotropin-releasing factor (CRF; CRF-BP) is strongly up-regulated in the tail of Xenopus tadpoles during spontaneous or T3-induced metamorphosis. This finding led us to investigate physiological roles for CRF and CRF-BP in tadpole tail. We found CRF, CRF-BP, and functional CRF1 receptor in tail and CRF and functional CRF1 receptors, but not CRF-BP, in the tail muscle-derived cell line XLT-15. CRF, acting via the CRF1 receptor, slowed spontaneous tail regression in explant culture and caused a reduction in caspase 3/7 activity. CRF increased, but stable CRF-BP overexpression decreased, [3H]thymidine incorporation in XLT-15 cells. Overexpression of CRF-BP in vivo accelerated the loss of tail muscle cells during spontaneous metamorphosis. Lastly, exposure of tail explants to hypoxia increased CRF and urocortin 1 but strongly decreased CRF-BP mRNA expression. We show that CRF is expressed in tadpole tail, is up-regulated by environmental stressors, and is cytoprotective. The inhibitory binding protein for CRF is regulated by hormones or by environmental stressors and can modulate CRF bioactivity.     

Thyroid hormone-dependent metamorphosis in a direct developing frog

The direct developing anuran, Eleutherodactylus coqui, lacks a tadpole, hatching as a tiny frog. We investigated the role of the metamorphic trigger, thyroid hormone (TH), in this unusual ontogeny. Expression patterns of the thyroid hormone receptors, TRalpha and TRbeta, were similar to those of indirect developers. TRbeta mRNA levels increased dramatically around the time of thyroid maturation, when remodeling events reminiscent of metamorphosis occur. Treatment with the goitrogen methimazole inhibited this remodeling, which was reinitiated on cotreatment with TH. Despite their radically altered ontogeny, direct developers still undergo a TH-dependent metamorphosis, which occurs before hatching. We propose a new model for the evolution of anuran direct development.     

Molecular and genetic studies suggest that thyroid hormone receptor is both necessary and sufficient to mediate the developmental effects of thyroid hormone

Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopuslaevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general.     

Multiple thyroid hormone-induced muscle growth and death programs during metamorphosis in Xenopus laevis

Xenopus laevis tadpole tails contain fast muscle fibers oriented in chevrons and two pairs of slow muscle "cords" along the length of the tail. When tail resorption is inhibited by a number of different treatments, fast muscle but not the slow cord muscle still is lost, demonstrating that the fast tail muscle is a direct target of the thyroid hormone-induced death program. Expression of a dominant negative form of the thyroid hormone receptor (TRDNalpha) was restricted to tadpole muscle by means of a muscle-specific promoter. Even though the transgene protects fast tail muscle from thyroid hormone (TH)-induced death, the tail shortens, and the distal muscle chevrons at the tail tip are degraded. This default pathway for muscle death is probably caused by the action of proteolytic enzymes secreted by neighboring fibroblasts. Non-muscle tissues that are sensitive to TH, such as the fibroblasts, are not protected by the transgene when it is expressed solely in muscle. If allowed to develop to metamorphosis, these transgenic animals die at the climax of metamorphosis before tail resorption has begun. Their limbs have very little muscle even though the rest of limb morphology is normal. Thus, fast tail muscle and limb muscle have their own cell autonomous death and growth programs, respectively, that are independent of the fate of the other neighboring cell types. In contrast, death of the slow muscle is controlled by the other cell types of the tail.     

Tumor necrosis factor-alpha attenuates thyroid hormone-induced apoptosis in vascular endothelial cell line XLgoo established from Xenopus tadpole tails

Amphibian metamorphosis induced by T(3) involves programmed cell death and the differentiation of various types of cells in degenerated and reconstructed tissues. However, the signaling pathway that directs the T(3)-dependent cell-fate determinations remains unclear. TNF-alpha is a pleiotropic cytokine that affects diverse cellular responses. Engagement of TNF-alpha with its receptor (TNFR1) causes intracellular apoptotic and/or survival signaling. To investigate TNF signaling functions during anuran metamorphosis, we first identified Xenopus laevis orthologs of TNF (xTNF)-alpha and its receptor. We found that xTNF-alpha activated nuclear factor-kappaB in X. laevis A6 cells through the Fas-associated death domain and receptor-interacting protein 1. Interestingly, xTNF-alpha mRNA in blood cells showed prominent expression at prometamorphosis during metamorphosis. Next, to elucidate the apoptotic and/or survival signaling induced by xTNF-alpha in an in vitro model of metamorphosis, we established a vascular endothelial cell line, XLgoo, from X. laevis tadpole tail. XLgoo cells formed actin stress fibers and elongated in response to xTNF-alpha. T(3) induced apoptosis in these cells, but the addition of xTNF-alpha blocked the T(3)-induced apoptosis. In addition, treatment of the cells with T(3) for 2 d induced the expression of thyroid hormone receptor-beta and caspase-3, and this thyroid hormone receptor-beta induction was drastically repressed by xTNF-alpha. Furthermore, in organ culture of the tail, xTNF-alpha significantly attenuated the tail degeneration induced by T(3). These findings suggested that xTNF-alpha could protect vascular endothelial cells from apoptotic cell death induced by T(3) during metamorphosis and thereby participate in the regulation of cell fate.     

Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis

Thyroid hormone (TH) induces the dramatic morphological and physiological changes that together comprise amphibian metamorphosis. TH-responsive tissues vary widely with developmental timing of TH-induced changes. How larval tadpole tissues are able to employ distinct metamorphic programs in a developmental stage- and TH-dependent manner is still unknown. Recently, several proteins capable of transporting TH have been identified. TH action and metabolism occurs primarily intracellularly, highlighting the importance of TH transporters. We examined the hypothesis that TH transporter expression and tissue distribution play an important role in mediating TH-induced metamorphic events. Xenopus tropicalis homologs for known TH transporting OATP, MCT and LAT family proteins were identified and gene specific qRT-PCR primers were developed. Total RNA was extracted from tissues representing three unique developmental fates including: growth/differentiation (hind limb), death/resorption (gill, tail) and remodeling (brain, liver, kidney). For growing and resorbing tissues, results showed the general trend of low initial expression levels of MCT8 and MCT10 transporters, followed by a several-fold increase of expression as the tissue undergoes TH-dependent metamorphic changes. The expression pattern in remodeling tissues was less uniform: a general decrease in transporter expression was observed in the liver, while the kidney and brain exhibited a range of expression patterns for several TH transporters. Collectively, these developmental expression patterns are consistent with TH transporting proteins playing a role in the effects of TH in peripheral tissues.     

Tadpole skin dies autonomously in response to thyroid hormone at metamorphosis

Transgenic tadpoles that express a dominant negative thyroid hormone (TH) receptor specifically in their skin undergo normal metamorphosis, with one exception: they retain a larval epidermis over the developing adult epithelium. TH-induced death of the tadpole epidermis is inhibited by the dominant negative TH receptor whereas the TH-induced response of the neighboring fibroblasts and the cells that form the adult skin occur normally. Therefore death of the tadpole skin is a direct and cell autonomous target of TH, and its protection has no detectable influence on TH-induced changes of other cell types.     

A balance between the diap1 death inhibitor and reaper and hid death inducers controls steroid-triggered cell death in Drosophila

The steroid hormone ecdysone directs the massive destruction of obsolete larval tissues during Drosophila metamorphosis, providing a model system for defining the molecular mechanisms of steroid-regulated programmed cell death. Although earlier studies have identified an ecdysone triggered genetic cascade that immediately precedes larval tissue cell death, no death regulatory genes have been functionally linked to this death response. We show here that ecdysone-induced expression of the death activator genes reaper (rpr) and head involution defective (hid) is required for destruction of the larval midgut and salivary glands during metamorphosis, with hid playing a primary role in the salivary glands and rpr and hid acting in a redundant manner in the midguts. We also identify the Drosophila inhibitor of apoptosis 1 as a survival factor in the larval cell death pathway, delaying death until its inhibitory effect is overcome by rpr and hid. This study reveals functional interactions between rpr and hid in Drosophila cell death responses and provides evidence that the precise timing of larval tissue cell death during metamorphosis is achieved through a steroid-triggered shift in the balance between the Drosophila inhibitor of apoptosis 1 and the rpr and hid death activators.     

Sea lamprey (Petromyzon marinus) contain four developmentally regulated serum thyroid hormone distributor proteins

Thyroid hormones (THs) are very lipophilic molecules which require a distribution network for efficient transport in serum. Despite observations that THs function in a wide variety of processes, including aspects of fish development (i.e., flat fish metamorphosis and smoltification), the proteins responsible for TH distribution in fish serum remain poorly studied. We chose to investigate the serum TH distributor proteins (THDPs) in lampreys. As one of only two extant agnathans, data on lamprey THDPs may offer new insights into the evolution of the vertebrate TH distribution network and serum proteins in general. Moreover, lampreys appear to contradict the vertebrate model of an increase in TH concentrations initiating and driving vertebrate metamorphosis. We show for the first time that sea lamprey serum contains at least four THDPs and that their presence in serum is temporally regulated throughout the life cycle. The albumin, glycoprotein AS is the dominant THDP present in the sera of larval and metamorphosing sea lamprey. In stage seven of metamorphosis, three additional THDPs appear, including the albumin, glycoprotein SDS-1; the glycolipoprotein CB-III; and an unidentified low molecular weight protein temporarily named Spot-5. The sera of parasitic and upstream migrant sea lampreys lack AS; their serum THDPs are SDS-1, CB-III, and Spot-5. Our data indicate that despite the change in type and number of THDPs, the overall total TH binding capacity of sea lamprey serum remains fairly stable until stage 7 of metamorphosis when a only modest decrease in total binding capacity is observed. Collectively these data indicate that the decline in serum TH concentrations observed during lamprey metamorphosis is not a consequence of a reduction in the distribution and storage capacity of the serum.     

Higher thyroid hormone receptor expression correlates with short larval periods in spadefoot toads and increases metamorphic rate

Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have (1) late onset of TR expression prior to the production of endogenous TH and (2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its higher TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.     

An antiapoptotic role of sorting nexin 7 is required for liver development in zebrafish

Sorting nexin (SNX) family proteins are best characterized for their abilities to regulate protein trafficking during processes such as endocytosis of membrane receptors, endosomal sorting, and protein degradation, but their in vivo functions remain largely unknown. We started to investigate the biological functions of SNXs using the zebrafish model. In this study, we demonstrated that SNX7 was essential for embryonic liver development. Hepatoblasts were specified normally, and the proliferation of these cells was not affected when SNX7 was knocked down by gene-specific morpholinos; however, they underwent massive apoptosis during the early budding stage. SNX7 mainly regulated the survival of cells in the embryonic liver and did not affect the viability of cells in other endoderm-derived organs. We further demonstrated that down-regulation of SNX7 by short interfering RNAs induced apoptosis in cell culture. At the molecular level, the cellular FLICE-like inhibitory protein (c-FLIP)/caspase 8 pathway was activated when SNX7 was down-regulated. Furthermore, overexpression of c-FLIP(S) was able to rescue the SNX7 knockdown-induced liver defect. CONCLUSION: SNX7 is a liver-enriched antiapoptotic protein that is indispensable for the survival of hepatoblasts during zebrafish early embryogenesis.     

Do all programmed cell deaths occur via apoptosis?

During development, large numbers of cells die by a nonpathological process referred to as programmed cell death. In many tissues, dying cells display similar changes in morphology and chromosomal DNA organization, which has been termed apoptosis. Apoptosis is such a widely documented phenomenon that many authors have assumed all programmed cell deaths occur by this process. Two well-characterized model systems for programmed cell death are (i) the death of T cells during negative selection in the mouse thymus and (ii) the loss of intersegmental muscles of the moth Manduca sexta at the end of metamorphosis. In this report we compare the patterns of cell death displayed by T cells and the intersegmental muscles and find that they differ in terms of cell-surface morphology, nuclear ultrastructure, DNA fragmentation, and polyubiquitin gene expression. Unlike the T cells, which are known to die via apoptosis, we find that the intersegmental muscles display few of the features that characterize apoptosis. These data suggest that more than one cell death mechanism is used during development.     

Caspase-9 regulates apoptosis/proliferation balance during metamorphic brain remodeling in Xenopus

During anuran metamorphosis, the tadpole brain is transformed producing the sensorial and motor systems required for the frog's predatory lifestyle. Nervous system remodeling simultaneously implicates apoptosis, cell division, and differentiation. The molecular mechanisms underlying this remodeling have yet to be characterized. Starting from the observation that active caspase-9 and the Bcl-X(L) homologue, XR11 are highly expressed in tadpole brain during metamorphosis, we determined their implication in regulating the balance of apoptosis and proliferation in the developing tadpole brain. In situ hybridization showed caspase-9 mRNA to be expressed mainly in the ventricular area, a site of neuroblast proliferation. To test the functional role of caspase-9 in equilibrating neuroblast production and elimination, we overexpressed a dominant-negative caspase-9 protein, DN9, in the tadpole brain using somatic gene transfer and germinal transgenesis. In both cases, abrogating caspase-9 activity significantly decreased brain apoptosis and increased numbers of actively proliferating cells in the ventricular zone. Moreover, overexpression of XR11 with or without DN9 was also effective in decreasing apoptosis and increasing cell division in the tadpole brain. We conclude that XR11 and caspase-9, two key members of the mitochondrial death pathway, are implicated in controlling the proliferative status of neuroblasts in the metamorphosing Xenopus brain. Modification of their expression during the critical period of metamorphosis alters the outcome of metamorphic neurogenesis, resulting in a modified brain phenotype in juvenile Xenopus.