Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis.

Thyroid hormones are crucial regulators of metamorphosis and development in bilaterians, particularly in chordate deuterostomes. Recent evidence suggests a role for thyroid hormone signaling, principally via 3,5,3',5'-Tetraiodo-L-thyronine (T4), in the regulation of metamorphosis, programmed cell death and skeletogenesis in echinoids (sea urchins and sand dollars) and sea stars. Here, we test whether TH signaling in skeletogenesis is a shared trait of Echinozoa (Echinoida and Holothouroida) and Asterozoa (Ophiourida and Asteroida). We demonstrate dramatic acceleration of skeletogenesis after TH treatment in three classes of echinoderms: sea urchins, sea stars, and brittle stars (echinoids, asteroids, and ophiuroids). Fluorescently labeled thyroid hormone analogues reveal thyroid hormone binding to cells proximal to regions of skeletogenesis in the gut and juvenile rudiment. We also identify, for the first time, a potential source of thyroxine during gastrulation in sea urchin embryos. Thyroxine-positive cells are present in tip of the archenteron. In addition, we detect thyroid hormone binding to the cell membrane and nucleus during metamorphic development in echinoderms. Immunohistochemistry of phosphorylated MAPK in the presence and absence of TH-binding inhibitors suggests that THs may act via phosphorylation of MAPK (ERK1/2) to accelerate initiation of skeletogenesis in the three echinoderm groups. Together, these results indicate that TH regulation of mesenchyme cell activity via integrin-mediated MAPK signaling may be a conserved mechanism for the regulation of skeletogenesis in echinoderm development. In addition, TH action via a nuclear thyroid hormone receptor may regulate metamorphic development. Our findings shed light on potentially ancient pathways of thyroid hormone activity in echinoids, ophiuroids, and asteroids, or on a signaling system that has been repeatedly co-opted to coordinate metamorphic development in bilaterians.

Thyroid hormone membrane receptor binding and transcriptional regulation in the sea urchin Strongylocentrotus purpuratus.

Thyroid hormones (THs) are small amino acid derived signaling molecules with broad physiological and developmental functions in animals. Specifically, their function in metamorphic development, ion regulation, angiogenesis and many others have been studied in detail in mammals and some other vertebrates. Despite extensive reports showing pharmacological responses of invertebrate species to THs, little is known about TH signaling mechanisms outside of vertebrates. Previous work in sea urchins suggests that non-genomic mechanisms are activated by TH ligands. Here we show that several THs bind to sea urchin (Strongylocentrotus purpuratus) cell membrane extracts and are displaced by ligands of RGD-binding integrins. A transcriptional analysis across sea urchin developmental stages shows activation of genomic and non-genomic pathways in response to TH exposure, suggesting that both pathways are activated by THs in sea urchin embryos and larvae. We also provide evidence associating TH regulation of gene expression with TH response elements in the genome. In ontogeny, we found more differentially expressed genes in older larvae compared to gastrula stages. In contrast to gastrula stages, the acceleration of skeletogenesis by thyroxine in older larvae is not fully inhibited by competitive ligands or inhibitors of the integrin membrane receptor pathway, suggesting that THs likely activate multiple pathways. Our data confirms a signaling function of THs in sea urchin development and suggests that both genomic and non-genomic mechanisms play a role, with genomic signaling being more prominent during later stages of larval development.

Thyroid hormone-induced cell death in sea urchin metamorphic development.

Thyroid hormones (THs) are important regulators of development, metabolism and homeostasis in metazoans. Specifically, they have been shown to regulate the metamorphic transitions of vertebrates and invertebrates alike. Indirectly developing sea urchin larvae accelerate the formation of juvenile structures in response to thyroxine (T4) treatment, while reducing their larval arm length. The mechanisms underlying larval arm reduction are unknown and we hypothesized that programmed cell death (PCD) is linked to this process. To test this hypothesis, we measured larval arm retraction in response to different THs (T4, T3, rT3, Tetrac) and assessed cell death in larvae using three different methods (TUNEL, YO-PRO-1 and caspase-3 activity) in the sea urchin Strongylocentrotus purpuratus. We also compared the extent of PCD in response to TH treatment before and after the invagination of the larval ectoderm, which marks the initiation of juvenile development in larval sea urchin species. We found that T4 treatment results in the strongest reduction of larval arms but detected a significant increase of PCD in response to T4, T3 and Tetrac in post-ingression but not pre-ingression larvae. As post-ingression larvae have initiated metamorphic development and therefore allocate resources to both larval and the juvenile structures, these results provide evidence that THs regulate larval development differentially via PCD. PCD in combination with cell proliferation likely has a key function in sea urchin development.

Evolution of non-genomic nuclear receptor function.

Nuclear receptors (NRs) are responsible for the regulation of diverse developmental and physiological systems in metazoans. NR actions can be the result of genomic and non-genomic mechanisms depending on whether they act inside or outside of the nucleus respectively. While the actions of both mechanisms have been shown to be crucial to NR functions, non-genomic actions are considered less frequently than genomic actions. Furthermore, hypotheses on the origin and evolution of non-genomic NR signaling pathways are rarely discussed in the literature. Here we summarize non-genomic NR signaling mechanisms in the context of NR protein family evolution and animal phyla. We find that NRs across groups and phyla act via calcium flux as well as protein phosphorylation cascades (MAPK/PI3K/PKC). We hypothesize and discuss a possible synapomorphy of NRs in the NR1 and NR3 families, including the thyroid hormone receptor, vitamin D receptor, ecdysone receptor, retinoic acid receptor, steroid receptors, and others. In conclusion, we propose that the advent of non-genomic NR signaling may have been a driving force behind the expansion of NR diversity in Cnidarians, Placozoans, and Bilaterians.

Thyroid Hormones Accelerate Initiation of Skeletogenesis via MAPK (ERK1/2) in Larval Sea Urchins (Strongylocentrotus purpuratus).

Thyroid hormones are important regulators of development and metabolism in animals. Their function via genomic and non-genomic actions is well-established in vertebrate species but remains largely elusive among invertebrates. Previous work suggests that thyroid hormones, principally 3,5,3',5'-Tetraiodo-L-thyronine (T4), regulate development to metamorphosis in sea urchins. Here we show that thyroid hormones, including T4, 3,5,3'-triiodo-l-thyronine (T3), and 3,5-Diiodothyronine (T2) accelerate initiation of skeletogenesis in sea urchin gastrulae and pluteus larvae of the sea urchin Strongylocentrotus purpuratus, as measured by skeletal spicule formation. Fluorescently conjugated hormones show T4 binding to primary mesenchyme cells in sea urchin gastrulae. Furthermore, our investigation of TH mediated skeletogenesis shows that Ets1, a transcription factor controlling initiation of skeletogenesis, is a target of activated (phosphorylated) mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)]. As well, we show that PD98059, an inhibitor of ERK1/2 MAPK signaling, prevents the T4 mediated acceleration of skeletogenesis and upregulation of Ets1. In contrast, SB203580, an inhibitor of p38 MAPK signaling, did not inhibit the effect of T4. Immunohistochemistry revealed that T4 causes phosphorylation of ERK1/2 in presumptive primary mesenchyme cells and the basal membrane of epithelial cells in the gastrula. Pre-incubation of sea urchin gastrulae with RGD peptide, a competitive inhibitor of TH binding to integrins, inhibited the effect of T4 on skeletogenesis. Together, these experiments provide evidence that T4 acts via a MAPK- (ERK1/2) mediated integrin membrane receptor to accelerate skeletogenesis in sea urchin mesenchyme cells. These findings shed light, for the first time, on a putative non-genomic pathway of TH action in a non-chordate deuterostome and help elucidate the evolutionary history of TH signaling in animals.

Evolution of thyroid hormone signaling in animals: Non-genomic and genomic modes of action.

Much research has focused on vertebrate thyroid hormone (TH) synthesis and their function in development and metabolism. While important differences in TH synthesis and signaling exist, comparative studies between vertebrates fail to explain the evolutionary origins of this important regulatory axis. For that, one needs to make sense out of the diverse TH effects which have been described in invertebrate phyla but for which a mechanistic understanding is largely missing. Almost every major group of non-vertebrate animals possesses the capability to synthesize and metabolize thyroid hormones and there is evidence for a nuclear thyroid hormone receptor mediated mechanism in the bilateria, especially in molluscs, echinoderms, cephalochordates and ascidians. Still, genomic pathways cannot fully explain many observed effects of thyroid hormones in groups such as cnidarians, molluscs, and echinoderms and it is therefore possible that TH may signal via other mechanisms, such as non-genomic signaling systems via membrane bound or cytoplasmic receptors. Here we provide a brief review of TH actions in selected invertebrate species and discuss the hypothesis that non-genomic TH action may have played a critical role in TH signaling throughout animal evolution.