The amphioxus genome enlightens the evolution of the thyroid hormone signaling pathway

Thyroid hormones (THs) have pleiotropic effects on vertebrate development, with amphibian metamorphosis as the most spectacular example. However, developmental functions of THs in non-vertebrate chordates are largely hypothetical and even TH endogenous production has been poorly investigated. In order to get better insight into the evolution of the thyroid hormone signaling pathway in chordates, we have taken advantage of the recent release of the amphioxus genome. We found amphioxus homologous sequences to most of the genes encoding proteins involved in thyroid hormone signaling in vertebrates, except the fast-evolving thyroglobulin: sodium iodide symporter, thyroid peroxidase, deiodinases, thyroid hormone receptor, TBG, and CTHBP. As only some genes encoding proteins involved in TH synthesis regulation were retrieved (TRH, TSH receptor, and CRH receptor but not their corresponding receptors and ligands), there may be another mode of upstream regulation of TH synthesis in amphioxus. In accord with the notion that two whole genome duplications took place at the base of the vertebrate tree, one amphioxus gene often corresponded to several vertebrate homologs. However, some amphioxus specific duplications occurred, suggesting that several steps of the TH pathway were independently elaborated in the cephalochordate and vertebrate lineages. The present results therefore indicate that amphioxus is capable of producing THs. As several genes of the TH signaling pathway were also found in the sea urchin genome, we propose that the thyroid hormone signaling pathway is of ancestral origin in chordates, if not in deuterostomes, with specific elaborations in each lineage, including amphioxus.     

Endogenous thyroid hormone synthesis in facultative planktotrophic larvae of the sand dollar Clypeaster rosaceus: implications for the evolutionary loss of larval feeding

Critical roles of hormones in metamorphic life history transitions are well documented in amphibians, lampreys, insects, and many plant species. Recent evidence suggests that thyroid hormones (TH) or TH-like compounds can regulate development to metamorphosis in echinoids (sea urchins, sand dollars, and their relatives). Moreover, previous research has provided evidence for endogenous hormone synthesis in both feeding and nonfeeding echinoderm larvae. However, the mechanisms for endogenous synthesis remain largely unknown. Here, we show that facultatively planktotrophic larvae (larvae that reach metamorphosis in the absence of food but have the ability to feed) from the subtropical sea biscuit Clypeaster rosaceus can synthesize thyroxine endogenously from incorporated iodine (I(125)). When treated with the goitrogen thiourea (a peroxidase inhibitor), iodine incorporation, thyroxine synthesis, and metamorphosis are all blocked in a dose-dependent manner. The inhibitory effect on metamorphosis can be rescued by administration of exogenous thyroxine. Finally, we demonstrate that thiourea induces morphological changes in feeding structures comparable to the phenotypic plastic response of larval structures to low food conditions, further supporting a signaling role of thyroxine in regulating larval morphogenesis and phenotypic plasticity. We conclude that upregulation of endogenous hormone synthesis might have been associated with the evolution of nonfeeding development, subsequently leading to morphological changes characteristic of nonfeeding development.     

The Control of Sea Urchin Metamorphosis: Ionic Effects

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus . At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation-free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.     

Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of nonfeeding development

Recent work on a diverse array of echinoderm species has demonstrated, as is true in amphibians, that thyroid hormone (TH) accelerates development to metamorphosis. Interestingly, the feeding larvae of several species of sea urchins seem to obtain TH through their diet of planktonic algae (exogenous source), whereas nonfeeding larvae of the sand dollar Peronella japonica produce TH themselves (endogenous source). Here we examine the effects of TH (thyroxine) and a TH synthesis inhibitor (thiourea) on the development of Dendraster excentricus, a sand dollar with a feeding larva. We report reduced larval skeleton lengths and more rapid development of the juvenile rudiment in the exogenous TH treatments when compared to controls. Also, larvae treated with exogenous TH reached metamorphic competence faster at a significantly reduced juvenile size, representing the greatest reduction in juvenile size ever reported for an echinoid species with feeding larvae. These effects of TH on D. excentricus larval development are strikingly similar to the phenotypically plastic response of D. excentricus larvae reared under high food conditions. We hypothesize that exogenous (algae-derived) TH is the plasticity cue in echinoid larvae, and that the larvae use ingested TH levels as an indicator for larval nutrition, ultimately signaling the attainment of metamorphic competence. Furthermore, our experiments with the TH synthesis inhibitor thiourea indicate that D. excentricus larvae can produce some TH endogenously. Endogenous TH production might, therefore, be a shared feature among sand dollars, facilitating the evolution of nonfeeding larval development in that group. Mounting evidence on the effects of thyroid hormones in echinoderm development suggests life-history models need to incorporate metamorphic hormone effects and the evolution of metamorphic hormone production.     

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.     

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.     

The history of a developmental stage: metamorphosis in chordates

Metamorphosis displays a striking diversity in chordates, a deuterostome phylum that comprises vertebrates, urochordates (tunicates), and cephalochordates (amphioxus). In anuran amphibians, the tadpole loses its tail, develops limbs, and undergoes profound changes at the behavioral, physiological, biochemical, and ecological levels. In ascidian tunicates, the tail is lost and the head tissues are drastically remodeled into the adult animal, whereas in amphioxus, the highly asymmetric larva transforms into a relatively symmetric adult. This wide diversity has led to the proposal that metamorphosis evolved several times independently in the different chordate lineages during evolution. However, the molecular mechanisms involved in metamorphosis are largely unknown outside amphibians and teleost fishes, in which metamorphosis is regulated by the thyroid hormones (TH) T3 and T4 binding to their receptors (thyroid hormone receptors). In this review, we compare metamorphosis in chordates and then propose a unifying definition of the larva-to-adult transition, based on the conservation of the role of THs and some of their derivatives as the main regulators of metamorphosis. According to this definition, all chordates (if not, all deuterostomes) have a homologous metamorphosis stage during their postembryonic development. The intensity and the nature of the morphological remodeling varies extensively among taxa, from drastic remodeling like in some ascidians or amphibians to more subtle events, as in mammals.     

Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus)

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.     

Active metabolism of thyroid hormone during metamorphosis of amphioxus

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.     

Metamorphosis in the Urodelan amphibians: patterns, regulation mechanisms, and evolution

Patterns of metamorphosis and mechanisms of its regulation in primitive and advanced salamanders are compared. It is found that urodelan evolution was characterised by the following trends: 1) increase in the number of metamorphosing systems; 2) increase in the amplitude of metamorphic transformations of each particular system due to the progressive divergence of the larval and the adult morphology; 3) synchronization of metamorphic transformations and their concentration within a relatively short period of ontogeny; 4) increase in the role of the thyroid hormones (TH) in the regulation of metamorphosis. Structures that are induced by factors other than TH and develop independently of TH in primitive Urodela species acquire TH-dependence in phyletically more advanced salamanders. For instance, morphogenetic induction as a mechanism of ontogeny regulation is substituted by endocrine induction with TH as the inducing factor. The switch from morphogenetic to endocrine induction stimulates the following events: 1) optimization of ontogeny; 2) reduction of the metamorphosis duration; 3) formation of the dissociability of larval and post-metamorphic stages of ontogeny, which, in its turn, is a precondition for the swith to necrobiotic metamorphosis and to the direct development.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.     

Expression of enzymes involved in thyroid hormone metabolism during the early development of Xenopus tropicalis

BACKGROUND INFORMATION: There are significant indications that amphibians require TH (thyroid hormones) prior to their involvement in the regulation of metamorphosis and before the development of a functional thyroid. RESULTS: In order to investigate the potential role for TH in pre-metamorphic Xenopus tropicalis we have cloned cDNAs for, and analysed the expression of, TPO (thyroid peroxidase), 5'DII (type II iodothyronine deiodinase) and 5DIII (type III iodothyronine deiodinase), enzymes involved in TH metabolism. Zygotic expression of TPO was detected in neurula stage embryos. Expression was observed in the notochord and later in the thyroid. The notochord was also a common site of expression for 5'DII and 5DIII. Other sites of 5'DII expression are the otic vesicles, retina, liver, blood-forming region, branchial arches and brain. 5DIII is also expressed in the brain, retina, liver, developing pro-nephros, blood-forming region and branchial arches. Embryos exposed to the TPO inhibitor methimazole showed a distinctive dose-dependent phenotype of a crimped notochord and shortened axis, together with alterations in (125)I(-) uptake. CONCLUSIONS: These data suggest a novel extrathyroidal role for TH during early development, and support the proposal that embryos require thyroid signalling for normal development prior to metamorphosis.     

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

Amphioxus postembryonic development reveals the homology of chordate metamorphosis

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.