Abstract
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.
Highlights
Echinoderms are the closest relatives to the vertebrates that produce a mineralized skeleton, though they independently evolved the capability for biomineralization [1]
T4 increased the rate of spicule formation in gastrulae and early pluteus larvae relative to both the control, and to the biologically inactive rT3 (Figure 3B; 3.1-fold in gastrulae, Wn = 48.7, p < 0.001; 2.3-fold in early stage larvae, Wn = 35.7, p < 0.001)
When gastrulae were exposed to two inhibitors of mitogen-activated protein kinase (MAPK) signaling, SB203580, and PD98059, both with and without added T4, we found that T4 induced skeletogenesis was inhibited by PD98059, but not by SB203580 (Figure 7)
Summary
Echinoderms are the closest relatives to the vertebrates that produce a mineralized skeleton, though they independently evolved the capability for biomineralization [1]. Sea urchins produce an extensive skeleton during both major life cycle phases: the benthic adult and the planktonic pluteus larva (Figure 1). Larval skeletogenesis (the process by which skeleton is formed de novo) appears to be regulated by a gene regulatory module that is responsible for juvenile and adult skeletogenesis [6]. This gene regulatory mechanism has been the focus of extensive research in the purple sea urchin Strongylocentrotus purpuratus and related species [for example, [7,8,9,10,11]]. A MAPK cascade phosphorylating the transcription factors Ets and Alx is necessary for skeletogenesis [Figure 2; [12]]
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