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

Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis Elias Taylor, Andreas Heyland This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3858209/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Developmental Biology Advances → Version 1 posted 8 You are reading this latest preprint version Abstract 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 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. 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 skeletogenesis in the three echinoderm groups. Additionally, we detect thyroid hormone binding to the cell membrane and nucleus during metamorphic development in echinoderms. 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. Additionally, 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Thyroid hormones (THs) regulate metamorphosis, skeletogenesis, and diverse physiological/developmental systems in chordate deuterostomes. Evidence also suggests a role for TH signaling in non-chordate bilaterians (Carpizo-Ituarte, 1993 ; Chino et al., 1994; Eales, 1997 ; Heyland et al., 2004 ; Holzer, 2015 ; Sainath et al., 2019 ; Saito et al., 1998 ; Taylor et al., 2023 ; Taylor & Heyland, 2017 ). Thyroxine (T4) is the most abundant TH analogue in nature. Many non-bilaterians use THs and other iodinated tyrosine derivatives as structural elements or to scavenge free radicals (reviewed in Taylor & Heyland, 2017 ). T4 is the typical first step in TH synthesis (Eales, 1997 ; Edmiston et al., 1993 ; Phatarphekar et al., 2013 ; Roche, 1952 ; Siuda & DeBernardis, 1973 ; Taylor & Heyland, 2017 ), a biochemical process predating metazoans (Heyland & Moroz, 2005 ; Phatarphekar et al., 2013 ; Taylor & Heyland, 2017 ). Therefore, the use of THs as a signaling agent to regulate development and metamorphosis can be considered a derived trait of some metazoans. L-triiodothyronine (T3), a metabolite of T4 and active hormone in vertebrates has been shown to regulate development and metamorphosis (Brtko, 2021 ), with T4 acting synergistically via non-genomic mechanisms (Das et al., 2010 ; Davis et al., 2016 ). In contrast, T4 has roughly ten-fold greater efficacy than T3 on regulation of development, settlement, and metamorphosis in some non-chordates, including sea urchins and molluscs (Chino et al., 1994; Huang et al., 2015 ; Taylor et al., 2023 ; Taylor & Heyland, 2017 ). There are three major categories of mechanism by which thyroid hormone signal transduction occurs: 1) Genomic signaling via a nuclear hormone receptor (Brent, 2012 ; Taylor & Heyland, 2017 ), 2) Extra-nuclear actions of a nuclear hormone receptor (Davis et al., 2016 ; Taylor & Heyland, 2022 ), and 3) Integrin-mediated MAPK phosphorylation cascade (Davis et al., 2016 ; Taylor & Heyland, 2017 ). The specific mechanisms governing TH actions in non-chordates remain largely unknown, despite new insights from echinoderms, mollusks, and cnidarians. Two main TH receptors, the nuclear thyroid hormone receptor (THR) and RGD-binding integrins are common to all bilaterians and all metazoans respectively (Taylor & Heyland, 2017 , 2022 ). T4 has been shown to accelerate metamorphic development in echinoderms and molluscs (Carpizo-Ituarte, 1993 ; Chino et al., 1994; Fukazawa et al., 2001 ; Heyland et al., 2004 , 2006 ; Hodin et al., 2001 ; Johnson, 1998 ; Johnson & Cartwright, 1996 ; Saito et al., 1998 ). Evidence suggests that these mechanisms may rely on the nuclear hormone receptor-mediated pathway (Huang et al., 2015 ; Taylor et al., 2023 ) and that THR may regulate apoptosis and skeletogenesis in sea urchins (Taylor et al., 2023 ; Wynen et al., 2022 ). Additionally, embryonic and larval sea urchin skeletogenesis may be regulated by the TH integrin-mediated pathway (Taylor & Heyland, 2018 ). Taylor and Heyland ( 2017 ) suggested that sea urchin skeletogenesis is regulated by thyroid hormones via a non-genomic integrin-mediated MAPK cascade triggered by direct TH binding to skeletogenic mesenchyme cells. Additionally, Wynen et al. ( 2022 ) demonstrated a role for THs regulating apoptosis in metamorphic development. However, it is unclear if TH signaling and its involvement in skeletogenesis is limited to S. purpuratus larvae, or if it can be generalized to other sea urchins and echinoderms. Indirect-developing echinoderms are characterized by a biphasic life cycle with a planktonic phase followed by settlement to the benthos and metamorphosis. Sea urchin, brittle star, and sea star development to settlement involves the production of juvenile structures in the pre-settlement larva, including skeletal elements (e.g. skeletal plates, spines, test, tube-foot rings in sea urchins). The actions of thyroid hormones on skeletogenesis in sea urchins have been of particular interest, as sea urchin larval skeletogenesis is a thoroughly-studied model system (Shashikant et al., 2018 ). In particular, the gene regulatory network (and transcription factors regulating skeletogenesis have been characterized (e.g. Davidson et al., 2002 ; Longabaugh, 2012 ; Mann et al., 2010 ; McIntyre et al., 2014 ; Rafiq et al., 2014 ). The development of adult skeleton in a coelom or rudiment adjacent to the gut appears to be an ancient and conserved component of echinoderm development (Isaeva & Rozhnov, 2022 ; Mooi & David, 1998 ) and is considered a shared characteristic of all extant echinoderms. In contrast, larval skeletogenesis is likely a synapomorphy of holothuroids (sea cucumbers), echinoids (sea urchins), and ophiuroids (brittle stars) (Fig. 1; Erkenbrack & Thompson, 2019 ). Crinoids (sea lilies) and asteroids (sea stars) do not feature larval skeletogenesis (Shashikant et al., 2018 ). Here we use “adult skeletogenesis” to refer to the development of skeletal elements slated to be incorporated into the juvenile echinoderm, post-settlement (Fig. 2). This skeletal development begins prior to settlement, during metamorphic development when skeleton is deposited by a population of mesenchyme cells with a distinct but similar GRN to the primary mesenchyme cells (PMCs) responsible for larval skeleton (Gao & Davidson, 2008 ; Shashikant et al., 2018 ). For instance, sea urchins develop spines, portions of the adult test, and skeletal rings supporting the tube feet (Heyland & Hodin, 2014 ). Much of this development occurs in the juvenile rudiment, a structure adjacent to the larval gut which contains most tissues of the developing juvenile echinoderm. Similarly, the larval ophiuroid, asteroid, and holothuroid produce extensive skeleton prior to metamorphosis, mainly in the juvenile rudiment. In Crinoids, an outgroup to the other extant echinoderms, the skeletonized stalk develops from a coelom adjacent to the gut, while the calyx (analogous to the echinoid test), begins developing from extra-coelomic spicules. In many echinoids and ophiuroids, larval skeleton supports the larval arms, important structures for feeding and defense. During embryonic development, either during gastrulation or immediately following gastrulation, these echinoids and ophiuroids develop larval skeleton from a population of mesenchyme cells separate from the adult/late mesenchymal cells. In holothuroids and ophiuroids, these cells derive from the tip of the archenteron or adjacent coeloms. In echinoids, the PMCs arise from small micromeres at the vegetal pole. From there, the cells migrate to the ventrolateral cluster and form the larval arms in echinoids and ophiuroids. The GRN governing skeletogenesis in these cells is distinct, although it shares a reliance on expression of Alx1 and Ets1, two key transcription factors in the skeletogenic GRN. Asteroid mesenchymal cells do not express Alx1 until expansion of the coeloms prior to rudiment development, likely accounting for the potential loss of skeletogenesis in larval asteroids (Koga et al., 2016 ). Larval skeletogenesis in ophiuroids, echinoids, and holothuroids is a result of a similar GRN to the adult skeletogenic GRN being expressed early in development by mesenchyme cells arising during gastrulation (Gao & Davidson, 2008 ). There are two hypotheses of the origin of larval skeletogenesis; the first posits a single origin of adult skeletogenesis followed by multiple origins of larval skeletogenesis and convergent evolution of larval skeleton. This is owing to the phylogenetic placement of ophiuroids (which produce larval skeleton) as sister group to asteroids (which do not), as well as dissimilarities in the ophiuroid and echinoid larval GRN. The second hypothesis suggests that regardless of the sometimes contentious phylogenetic placement of ophiuroids (Cannon et al., 2014 ; Pisani et al., 2012 ; Reich et al., 2015 ; Telford et al., 2014 ), a single origin of larval skeletogenesis is more likely (Erkenbrack & Thompson, 2019 ), followed by a potential secondary loss of larval skeleton in Asteroidea. Echinoderm skeletogenic GRNs share specific characteristics, such as a conserved role for alx1, ets1, and vegfr, suggesting that differences in echinoid larval skeletogenesis and cell specification may be derived traits (Dylus et al., 2016 ; Erkenbrack & Thompson, 2019 ; McCauley et al., 2012 ). Both hypotheses indicate that adult skeletogenesis arose first, with a single origin. Given the shared origin of larval and adult skeletogenesis in echinoderms, and thyroid hormone regulation of larval and adult skeletogenesis in sea urchins (Taylor et al., 2023 ; Taylor & Heyland, 2018 ), we predict that TH regulation of skeletogenesis would be a shared feature of both larval and adult skeletogenesis in echinoids, ophiuroids, and asteroids. In this study, we seek to answer the question of whether thyroid hormones accelerate early and late skeletogenesis in two distantly related Echinoderms: the sea star P. ochraceus , and the brittle star O. aculeata , as well as to confirm previous results showing that thyroid hormones accelerate skeletogenesis in echinoids. We show that THs are capable of accelerating skeletogenesis in three classes of echinoderms, sea urchins, sea stars, and brittle stars (echinoids, asteroids, and ophiuroids). THs bind to cells proximal to sites of skeletogenesis and induce MAPK phosphorylation and skeletogenesis. These processes can be fully or partially inhibited by a competitive ligand of RGD-binding integrins, and by an inhibitor of MAPK activity. We discuss our findings in the context of the origin and evolution of TH regulation and the evolution of skeletogenesis in echinoderms. 2. Methods 2.1 Animal Care Ophiopholis aculeata were collected in Cobscook Bay, Maine, and shipped to Williamsburg, VA to be spawned (2020–2021). Embryos were shipped overnight on ice to the University of Guelph (ON, Canada) as fertilized eggs in the case of experiments on gastrulae, or 6 days post-fertilization (dpf) in the case of the late-larval experiments. On arrival, ophiuroid larvae were transferred to 2L beakers at a density of 0.5-1 larva/mL. The cultures were cleaned manually and had the water replaced with filtered (0.2 µm) and UV-treated fresh artificial seawater (Instant Ocean) three times weekly. At the same time, cultures were fed Rhodomonas salina (UTEX LB 2763) at a density of 2000 cells/mL and Dunaliella tertiolecta (UTEX LB 999) at 3000 cells/mL. Gastrulae were collected for skeletogenesis assays at 2–3 dpf. Late pluteus larvae were collected for skeletogenesis assays at 27 dpf. We collected Adult Pisaster ochraceus by hand during low tide at Friday Harbor, Washington (2019) and maintained them in seawater tables under flow-through seawater conditions (Hodin et al., 2019 ). After spawning and fertilization, hatched embryos were transferred to 2L jars at a density of 1 larva per 2 mL. The cultures were cleaned manually and had the water replaced with fresh seawater three times weekly. At the same time, cultures were fed Rhodomonas sp. at a density of 3000 cells/mL and Dunaliella sp. at 4000 cells/mL. Gastrulae were collected after 48 hours at 11–13°C and staged to ensure they were at early gastrulation (after ingression of primary mesenchyme cells and invagination of the blastopore, but before ingression of secondary mesenchyme cells and archenteron contact with the animal pole; see McClay et al., 2020 for review of gastrulation). Early bipinnaria were collected after 12 dpf, after coelomic pouch elongation. Late bipinnaria were collected at 22 dpf, just before formation of the brachiolaria arm buds. Adult urchins ( Strongylocentrotus purpuratus ) were shipped from Monterey, CA (2019–2023), where they were collected by diving and subsequently kept in tanks of artificial seawater at the Hagen Aqualab (University of Guelph, ON). The adults were fed a diet of kelp ( Macrocystis pyrifera , and Kombu spp .) every 2–3 days. Temperature was maintained at 14°C and salinity at 31 ppt). Urchins were spawned by injecting 0.5–1.5 mL of 0.5 M KCl in distilled water, depending on the size of the urchin. Sperm was collected dry by pipetting sperm directly from the gonopores. Females were inverted over a beaker of filtered artificial seawater (0.2µm – FASW) to collect eggs. After spawning, eggs were washed twice with FASW. Diluted sperm (approx. 1:100) was titrated into the beaker of eggs until fertilization success reached at least 90%. Fertilized eggs were washed once more with FASW to remove excess sperm and allowed to develop at 12°C in a 2 L beaker until hatching. After 48 hours at 12–14°C, hatched embryos were transferred to 2 L beakers at a density of 1 larva/mL. Sea urchin larval cultures were maintained at 14°C with salinity at 31–33 g/L and larval density was gradually reduced on a weekly basis to an eventual density of approximately 250 larvae per liter. Cultures were stirred constantly using a paddle system previously described by Strathman and Strathman (1983) and kept on a 12:12 light cycle. The cultures were cleaned manually and had the water replaced three times weekly. At the same time, cultures were fed Rhodomonas salina at a density of 3000 cells/mL and Dunaliella tertiolecta at 4000 cells/mL. Dendraster excentricus (sand dollars) were collected from Crescent Beach, East Sound, Orcas Island in 2019. D. excentricus were spawned by injecting 0.2-1.0 mL of 0.5 M KCl in distilled water, depending on the size of the animal. Sperm was collected dry by pipetting sperm directly from the gonopores. Females were inverted over a beaker of natural seawater to collect eggs. After spawning, eggs collected by pipette and distributed in a monolayer in a 500 mL beaker of seawater. Diluted sperm (approx. 1:100) was titrated into the beaker of eggs until fertilization success reached at least 90%. Fertilized eggs were washed once with seawater to remove excess sperm and allowed to develop at 11–13°C in a 500 mL beaker until hatching. After 20 hours at 14°C, hatched embryos were transferred to 2L jars at a density of 1 larva/mL. Gastrulae were collected shortly thereafter and staged to ensure they were at early gastrulation. 2.2 Hormone preparation Thyroid hormones (rT3, T4, T3) were prepared as described in Taylor and Heyland 2018 (dissolved in filtered artificial seawater with 1% DMSO at a working concentration of 10 − 3 M). PD98059 (inhibitor of MAPK ERK1/2; Sigma-Aldrich P215) and RGD peptide (inhibitor of integrin RGD binding pocket; Sigma-Aldrich A8052) were also used, and similarly diluted in filtered artificial seawater to a working concentration of 10 − 3 M. Larvae were exposed to a final concentration of 10 − 7 M rT3, T4, T3, or RGD peptide, or to 5 x 10 − 5 M PD98059. 2.3 Skeletogenesis assays Skeletogenic assays were conducted as described in Taylor and Heyland ( 2018 , 2023). We scored the presence of skeletal spicules in repeated measures of individually isolated live larvae and determined the proportion of individuals which had developed skeleton (hourly for gastrulae, daily for late-stage larvae). Presence of skeletal spicules was confirmed with polarized light microscopy. Experiments were considered complete when at least one group asymptotically reached skeletogenesis (over 95% of at least one group achieved skeletogenesis), typically over the course of 4 hours for gastrulae, or 3–5 days for late-stage larvae (S.p. gast.: 4 hr, S.p. larvae:, P.o. gast.: 72 hr, P.o. larvae: 120 hr, D.e. gast.: 4 hr, O.a.: gast: 5 hr, O.a. larvae: 96 hr). Larvae were kept at a density of 1 larva/mL in individual wells (24 well plate, 1mL/well). Larvae were placed on a slide for observation and skeletal structures were detected by polarized light microscopy on a compound microscope with inserted polarizing filter. Echinoderm skeletons are composed of a single birefringent crystal and polarized light microscopy is extremely suitable for detection of even initial skeletal spicules. In the case of gastrulae, skeleton was detected and scored at the ventrolateral clusters. In the case of late-stage larvae, the presence of adult skeleton developing in and around the rudiment was scored. Any skeleton which was not part of the larval arms was considered to be adult skeleton (Heyland & Hodin, 2014 ). 2.4 Fluorescent Microscopy Fluorescently labeled thyroxine (RHT4) was synthesized as described in Taylor and Heyland ( 2018 ) and used to determine T4 binding locations. RHT4 was prepared at a stock concentration of 10 − 3 M and diluted in PBS to a final concentration of 10 − 6 M for staining fixed samples. M8159 (antibody for phosphorylated ERK1/2; mouse monoclonal, Sigma-Aldrich M8159) was used to quantify MAPK (ERK1/2) phosphorylation (1:250). Hoechst 33342 (working stock 1 mg/mL, final concentration 5 µg/mL) was used to stain and identify nuclei. Thyroid hormones were diluted in filtered artificial seawater with DMSO (5:95 DMSO:FASW) to a working stock concentration of 10 − 3 M before being diluted to a final concentration of 10 − 7 M in exposure conditions. Larvae or gastrulae were exposed to 100 nM T4, 500 µM PD98059, 100 nM RGD peptide, 100 nM rT3, 100 nM RGD + 100 nM T4 or a vehicle control (FASW with 0.0005% DMSO) for 90 minutes. Subsequently, they were fixed in 2% formaldehyde for 10 minutes at room temperature and washed with methanol and placed for 5 minutes in a -20°C freezer. Samples were washed 3x with phosphate-buffered saline with 0.1% Tween-20 (PBST; 5 minutes between washes) at room temperature, and blocked with 1% Goat Serum in PBST for 60 mins. Samples were exposed to primary MAPK antibody (M8159 1:250) for 12 hours overnight at 4° C. They were then washed 5x in PBST (5 minutes between washes) and exposed to secondary antibody for 4 hours (1:1000). This was followed by 7x washes in PBS (5 minutes between washes). Finally, samples were exposed to a mixture of Hoechst 33342 (5 µg/mL) and RHT4 (10 − 6 M) in PBS for 15 minutes, followed by 3x washes in PBS and were then mounted for imaging in DABCO/glycerol (see similar protocol in Wynen et al., 2022 ). As the previously reported magnitude of TH effects on MAPK phosphorylation was over 3-fold increase in intensity (Taylor & Heyland, 2018 ), a sample size of 4–6 confocal images was predicted to be sufficient statistical power to test the effect of T4 on MAPK phosphorylation. 2.5 Statistical analysis MAPK phosphorylation was quantified by automatic cell counting in the Hoechst 33342 channel, followed by a measurement of fluorescent intensity of each individual cell in a radius around each nucleus (see S1 for details and S5 for ImageJ script). Intensity was normalized to the Hoechst 33342 signal to account for attenuation (Figure S1 ). Fluorescent intensity of the top 5% of cells was compared between experimental groups and the control by one-way ANOVA followed by Tukey’s post-hoc test. Intensities and means are reported as ± standard error. Colocalization of Hoechst nuclear stain, MAPK phosphorylation, and T4-binding locations in juvenile rudiments and midgut in late-stage larvae was measured with Pearson’s Coefficient using JACoP (v2.0; Bolte & Cordelières, 2006 ). For the skeletogenic assays, experimental groups were compared with binary logistic regression of skeletal presence (binomial) and time (hours or days) contrasted to the control group. Additionally, spicule proportion over time was transformed to a rate of skeletogenesis (spicules/hour or spicules/day) and compared to the control with one-way ANOVA followed by Tukey’s post-hoc. Proportions and means are reported as ± standard error. 3. Results 3.1. Thyroid hormone effects on skeletogenesis Thyroid hormones (T4, T3) accelerate skeletogenesis in a sea urchin ( Strongylocentrotus purpuratus ), a brittle star ( Ophiopholis aculeata ), and a sea star ( Pisaster Ochraceus ) (Fig. 3), as well as a sand dollar ( Dendraster excentricus ; Figure S1 and as previously shown in Heyland and Hodin 2004). However, THs were not found to induce skeletogenesis in Pisaster ochraceus (sea star) gastrulae (n = 20, ANOVA not possible as no groups produced skeleton; p = 1.00). THs, including T4 and T3, accelerated onset of juvenile skeletogenesis (n = 6, F(7,40) = 36.25, p < 0.001) in sea star bipinnaria. The effect of THs on sea star bipinnaria was inhibited by PD9809 and RGD peptide (Tukey’s HSD, p < 0.001). THs, including T4 and T3, accelerate skeletogenesis in O. aculeata gastrulae (n = 20, F(7,152) = 12.45; p < 0.001; Tukey’s HSD, T4: 95%CI[1.75-fold, 2.69-fold], T3: 95%CI[1.08-fold, 2.03-fold]). RGD peptide (RGD) and PD98059 (PD) partially inhibit the effects of THs on skeletogenesis (Tukey’s HSD, p < 0.001). In O. aculeata larvae, THs (T4) accelerated juvenile skeletogenesis (n = 12, F(3,44) = 24.22; Tukey’s HSD p < 0.001). This effect was partially inhibited by PD98059 (p = 0.0163). The effect of RGD peptide on skeletogenesis was not tested in late-stage O. aculeata . In all cases, T4 at 10 − 7 M had a stronger effect than T3 at 10 − 7 M (1.22-fold [S.p. 8-armed pluteus] to 3.71-fold [S.p. gastrula], x̄ = 2.31-fold). This difference was statistically significant in all cases except late-stage S. purpuratus (8-armed pluteus stage; Tukey’s HSD, alpha = 0.05). Gastrulae exposed to T4 showed rapid skeletogenesis (< 5h; Fig. 3M) in the ventrolateral clusters (Fig. 3A-B, E-F) in sea urchin and brittle stars, but not sea stars (Fig. 3I-J). This acceleration of skeletogenesis occurred over the course of 5 hours observation, shifting the onset of skeletogenesis forward by 1.05 h (± 0.07 h) in S. purpuratus (sea urchin) gastrulae and 1.12 h (± 0.13 h) in O. aculeata gastrulae. This represents a significant increase in the rate of skeletogenesis in T4-exposed gastrulae ( S. purpuratus : 3.13-fold, ± 0.26; O. aculeata : 2.22-fold, ± 0.31). THs accelerated skeletogenesis in older larvae of all three classes of Echinoderms (sea urchin, sea star, and brittle star; Fig. 3N). Sea urchin larvae displayed increased skeletogenesis in the developing rudiment, as well as extra-rudiment skeleton such as juvenile spines and genital plates. Over the course of seven days of observation, T4-exposed larvae developed skeleton in the juvenile rudiment 2.78 days in advance of the control group (1.70-fold increased rate, ± 0.14) and had an average rudiment skeletal development stage of 8.4 compared to the control group at 0.7 (staging scheme from Heyland & Hodin, 2014 ). T4 accelerated juvenile skeletogenesis of brittle star ophioplutei (23 d) in the terminal and radial plates adjacent to the gut and somatocoel (Fig. 3H). T4 brought forward the mean initiation of juvenile skeletogenesis by 0.96 d, resulting in a 1.79-fold (± 0.29) increase in the rate of skeletogenesis relative to the control. At the end of observation (27 d, 4 d post-exposure), every larva had begun juvenile skeletogenesis. T4 drastically accelerated skeletogenesis of sea star late bipinnaria/early brachiolaria larvae (24 d). After 24 hours of T4 exposure, P. ochraceus bipinnaria larvae produced skeletal spicules (oral plates) adjacent to the gut and somatocoel (Fig. 3L). By day 3, every single T4-exposed larva had produced skeleton, including oral and madreporitic plates, while not a single control larva had begun skeletogenesis (Fig. 3K). At the end of observation (29 d, 5 d post-exposure), 5/30 (± 2.7) of control larvae had produced skeleton. This represents a 4-day acceleration of skeletogenesis by T4, and a 22.6-fold (± 5.7) increase in the rate of skeletogenesis over the observed period. 3.2. Thyroid hormone signaling mechanisms RGD peptide (a competitive inhibitor of the RGD-binding pocket on RGD-binding integrins) inhibited T4 acceleration of larval skeletogenesis in D. excentricus (Figure S4 ), S. purpuratus (Fig. 4A), and O. aculeata gastrulae (Fig. 4C), as well as juvenile skeletogenesis in P. ochraceus late bipinnaria (Fig. 4E; binomial logistic regression, n = 12–40, p < 0.05). PD98059, an inhibitor of MAPK phosphorylation (ERK1/2), inhibited the effect of T4 on skeletogenesis in S. purpuratus and O. aculeata gastrulae (Fig. 4AC), as well as on juvenile skeletogenesis in P. ochraceus , S. purpuratus , and O. aculeata late-stage larvae (Fig. 4D-F; binomial logistic regression, n = 12–40, p < 0.05). P. ochraceus gastrulae did not produce skeleton either with or without thyroid hormone exposure and as such there were no differences in rate of skeletogenesis between any P. ochraceus gastrulae treatment groups (Fig. 4B). 3.3. T4 binding locations and colocalization with induced MAPK phosphorylation Thyroid hormone binding increased MAPK phosphorylation in S. purpuratus gastrulae (n = 4, p < 0.05). RGD peptide alone had no statistically significant effect on MAPK phosphorylation, but did inhibit the effect of T4 (n = 4, p < 0.05). THs (T4) bound to PMCs in sea urchin gastrulae (Fig. 5B), as well as to regions near the tip of the developing archenteron. MAPK activation was seen most strongly in secondary mesenchyme cells (neural precursors) and the coelomic pouches (rudiment precursors), as well as to a lesser degree in PMCs ingressing at the vegetal pole. Both MAPK activation and T4 binding locations appeared to be localized to the membrane and cytoplasm (Fig. 5). Typically, the site of MAPK phosphorylation was colocalized with T4 binding sites within the cell (Fig. 5C′′-C‴, D′′-D‴) in the SMCs and PMCs, but not in the coelomic pouches. We did not detect any clear signs of nuclear binding in our confocal images. T4 did not significantly increase MAPK phosphorylation in P. ochraceus gastrulae (t[8] = 0.526, p = 0.61; Fig. 6E). RGD peptide decreased MAPK phosphorylation (t[8] = 2.80, p = 0.023; Fig. 6E). No distinct T4 binding sites were detected in either the control (Fig. 6A,C) or T4-exposed larvae (Fig. 6B,D). MAPK phosphorylation was most active in the developing coelomic pouches and was asymmetrical in all samples (20/20). T4 bound primarily to the midgut, hindgut, and somatocoel in sea star bipinnaria (Fig. 7) and brachiolaria (Fig. 8). T4-exposed larvae displayed higher levels of MAPK phosphorylation in both bipinnaria and brachiolaria (t(10) = 3.42, p = 0.007; Fig. 8B; P. ochraceus ). In contrast, RGD peptide exposure resulted in a decrease in MAPK phosphorylation as well as inducing whole-body muscle contractions (Figure S2 ). Larvae exposed to both RGD peptide and T4 displayed no statistically significant difference in MAPK phosphorylation from the control group (t(10) = 1.31, p = 0.218). In bipinnaria, the region of strongest MAPK phosphorylation was the somatocoel (Fig. 7). T4 bound most strongly to the membrane of gut cells, predominantly at the cell-cell junctions, but also to the nucleus (Fig. 7C″). MAPK phosphorylation in the gut was strongly colocalized to cells which bind T4 (Fig. 7C″’). Few presumptive T4 binding sites were observed in the hydrocoel, although the adjacent midgut did bind T4 (Fig. 7B). We observed extensive MAPK phosphorylation and increased T4 binding-sites in older sea star brachiolaria (Fig. 8). Putative T4-binding cells in the hindgut adjacent to the extending somatocoel were numerous and intensely fluorescent. Unlike in the bipinnaria, we observed T4 binding sites in the hydrocoel. As well, T4-binding sites were occasionally observed in the membrane/cytoplasm but frequently colocalized well with nuclear stains (Fig. 8D,E; r̄(4) = 0.715). We found that sea star gut displayed highly asymmetrical putative T4 binding locations, with increased quantity and intensity of binding sites in the midgut and foregut closer to the somatocoel (Fig. 10). We observed possible T4-binding sites predominantly in the basal cell membrane of gut epithelia, as well as the membrane of gut-adjacent mesenchyme cells. Many of these cells also display binding in the nucleus, albeit to a lesser degree (r̄ = 0.566). There are more T4 binding cells adjacent to the somatocoel and the intensity of T4-binding appears to be higher adjacent to the somatocoel. In T4-exposed larvae, these locations colocalize approximately with increased MAPK phosphorylation in the gut, and to a lesser degree, the somatocoel and mesenchyme cells adhering to the gut/somatocoel boundary (t(10) = 3.594, p = 0.0049; Fig. 10B) relative to control larvae (Fig. 10A). RGD peptide prevents the effect of T4 on MAPK phosphorylation (t(10) = 3.16, p = 0.010), but potentially increases the number of T4-binding locations we observed (p = 0.023; Fig. 10C). In late-stage ophiuroid larvae (eight-armed ophiopluteus), T4 bound to the gut, somatocoel, and presumptive skeletogenic mesenchyme in posterolateral arms (Fig. 9). In the posterolateral arms, we detected T4 binding sites predominantly in mesenchymal cells adhering to the skeletal rod (Fig. 9D). Most skeleton-adhering T4-binding cells displayed increased MAPK phosphorylation in response to T4-exposure. T4 significantly increased MAPK phosphorylation in O. aculeata larval arms and somatocoel (t(6) = 2.594, p = 0.041; Fig. 9C). The effect of T4 on MAPK phosphorylation is potentially inhibited by RGD peptide with a mean decrease of 17% MAPK phosphorylation fluorescent intensity, although this difference was not statistically significant (t(6) = 1.08, p = 0.320; Fig. 9C). We imaged larvae with a variety of hydrocoel developmental stages, from the beginning of metamorphic development until the point at which the hydrocoel begins to wrap around the gut. A representative T4-exposed larva at the relatively advanced 5-lobed stage is depicted in Fig. 9 (B,E). No larvae examined displayed a high degree of T4-binding in the hydrocoel. In contrast, the somatocoel of most larvae presented with presumptive T4-binding sites, and T4-exposed samples showed a greater intensity of MAPK phosphorylation in the somatocoel, colocalized with T4-binding sites (Fig. 9F; r̄(6) = 0.748). In sea urchin ( S. purpuratus ) eight-armed pluteus larvae (rudiment soft tissue stages v-vii; Heyland & Hodin, 2014 ) RHT4 bound and fluoresced to gut cells, indicating potential T4-binding sites (as in the other echinoderms we examined). Additionally, T4 bound to the rudiment; primarily to the rapidly-developing layer most distal to the gut. In T4-exposed larvae, MAPK phosphorylation was induced in the gut and rudiment, strongly colocalized with putative T4-binding sites (r̄(6) = 0.862). A close examination of binding sites in the T4-exposed larvae showed T4 binding in both the nucleus and cell membrane (Fig. 10B). Nuclear binding was primarily observed in the rudiment (r̄(6) = 0.847), compared to the gut (r̄(6) = 0.459). This is in contrast with S. purpuratus gastrulae and control late-stage plutei which both showed binding primarily in the cell membrane and cytoplasm (Fig. 5B-D). 4. Discussion We found that THs (T4 and T3) accelerated skeletogenesis in larvae of distantly related echinoderm groups (sea stars, brittle stars, and sea urchins), suggesting a conserved regulatory mechanism of skeletogenic mesenchyme by THs. T4 bound to cells near sites of skeletogenic activity and increased MAPK phosphorylation. Both the acceleration of skeletogenesis and the MAPK phosphorylation was inhibited by RGD peptide (a competitive inhibitor of RGD-binding integrins), as well as PD98059 (an inhibitor of MAPK [ERK1/2] phosphorylation) in all species. These results provide further evidence for a role of nongenomic TH signaling via an integrin membrane receptor-mediated MAPK cascade and suggests a conserved regulatory mechanism between these groups. On a subcellular level, THs bind to the membrane and nucleus in echinoderms. We found nuclear binding to be more prominent in older echinoderm larvae, especially in P. ochraceus brachiolaria midgut and S. purpuratus rudiment nuclei. This coincides with evidence suggesting genomic transcriptional regulation is much more prominent in sea urchin eight-armed plutei relative to gastrulae (Taylor et al., 2023 ) and reports that TH levels rise in older larvae as they develop to metamorphosis (Chino et al., 1994a ). THs accelerate sea urchin rudiment development (Chino et al., 1994a ; Heyland et al., 2004 ; Taylor & Heyland, 2018 ), suggesting that the rudiment may be a primary site of genomic TH signaling and regulation of metamorphosis. We found that sea star bipinnaria showed the greatest acceleration of skeletogenesis by thyroid hormones in the late larval stages, with THs inducing skeletogenesis weeks in advance of typical development (e.g. Pia et al., 2012 ). This might be attributed to the early presence of coelomic mesenchyme adjacent to the gut, relative to S. purpuratus and O. aculeata , where late mesenchymal cells responsible for skeletogenesis don’t arise until rudiment formation (Gao & Davidson, 2008 ). Asymmetric binding of thyroid hormones to gut cells is common to sea urchins, sea stars, and brittle stars. This may reflect the asymmetric development of adult structures in pre-metamorphic echinoderms. A greater quantity and intensity of the putative binding sites in the gut wall nearest the somatocoel suggests a potential shared mechanism of signaling from the gut wall to developing adult structures in the early rudiment. TH exposure resulted in acceleration of skeletogenesis in the rudiment, a process blocked by inhibitors of TH binding to RGD-binding integrins. This provides preliminary evidence that TH signaling via a membrane integrin receptor in gut and rudiment cells may regulate skeletogenesis in adjacent mesenchyme. Another possibility is that THs in the gut are binding to membrane transporters. Exogenous TH uptake has been proposed for echinoderms (Eales, 1997 ; Heyland & Moroz, 2005 ; Miller & Heyland, 2013 ), and gut wall transporters would be a crucial element in exogenous hormone uptake (Miller & Heyland, 2010 ). The transporter hypothesis would partially explain why THs bind to some non-mesenchymal cells. These leave us with several explanatory hypotheses for this phenomenon: either THs are binding to transporters in the gut, or to receptors in the gut triggering release of a secondary signal (likely endocrine or neural, e.g. VEGF or serotonergic neuronal signaling). The arrangement of sea urchin hydrocoel and somatocoel is also unique within echinoderms. Sea urchin hydrocoel is layered on the somatocoel, while in most echinoderms the hydrocoel and somatocoel develop in distinct regions adjacent to the gut, with the hydrocoel developing proximal to the mouth region and the somatocoel developing closer to the midgut. In sea urchins, both somatocoel and hydrocoel develop adjacent to the midgut (M. M. Smith et al., 2008 ; Wessel et al., 2014 ). This rearrangement of developing tissues in sea urchins may be related to the early skeletal development in the hydrocoel and the presence of TH binding sites and acceleration of development of sea urchin hydrocoel by THs. Sea urchin hydrocoel forms skeleton during rudiment development, but sea star and brittle star hydrocoel does not. Similarly, THs bind to sea urchin hydrocoel; particularly to the tips of the developing tube feet where skeleton will form. T4 shows little to no binding to sea star and brittle star hydrocoel. This suggests a possible link between TH receptor expression in sea urchin rudiment hydrocoel and TH regulation of skeletogenesis in sea urchin rudiment, which may be a synapomorphy of sea urchins relative to other echinoderms. Additionally, T4 activates skeletogenesis in P. ochraceus before the coelomic sacs have wrapped around the gut, implying the source of skeletogenic cells is either migratory mesenchymal cells or gut cells, rather than the coelomic sac which will eventually form the hydrocoel. Cocurullo et al. ( 2023 ) provided circumstantial evidence based on transcriptional data that in sea urchin pluteus larvae, THs may be synthesized in the gut and neurons proximal to the gut (putative TH synthesis enzymes colocalized with typical gut and neuronal markers). They found expression of peroxidasin and deiodinase (both are potentially involved in TH synthesis), putative TH transporters, and both THR and integrin membrane receptor in early pluteus larvae. We found that the putative TH synthesis enzymes were regulated by THs in pre-metamorphic sea urchin larvae (Taylor et al., 2023 ). This corresponds well with previous models in which THs were both exogenously and endogenously sourced; consumed as part of a typical algal diet and synthesized by iodinating dityrosine residues (Eales, 1997 ; Heyland & Moroz, 2005 ). In both cases, THs would be sourced proximal to the gut. Given the lack of a circulatory system and hypothalamic-pituitary-thyroid axis, we predicted that TH binding sites in Echinoderm larvae might also be gut-proximal. As the juvenile rudiment is adjacent to the gut in species of Echinoderm which produce one, this would provide a plausible explanation for thyroid hormone regulation of development to metamorphosis, and settlement. 4.1. Evolution of skeletogenic gene regulation in echinoderms While the gene regulatory network (GRN) underlying adult skeletogenesis is an apparent apomorphy shared by extant echinoderms, larval skeletogenesis is likely a synapomorphy of sea stars, brittle stars, and sea urchins. (Shashikant et al., 2018 ). A possible model for the evolution of echinoderm larval skeletogenesis is of a single transfer of the adult GRN to early larval echinoderm development, followed by a secondary loss of larval skeleton in sea stars, and partial loss in sea cucumbers (Erkenbrack & Thompson, 2019 ). Alternatively, multiple origins of skeletogenesis in Echinodermata have been proposed and this hypothesis remains under investigation (Cary & Hinman, 2017 ; Dylus et al., 2016 ; McCauley et al., 2012 ). MAPK phosphorylation of Ets1 is responsible for the majority of MAPK effects in skeletogenic sea urchin mesenchyme (Shashikant et al., 2018 ), and a MAPK cascade phosphorylating Ets1 is necessary for skeletogenesis in sea urchin larvae (Fernandez-Serra et al., 2004 ; Khor & Ettensohn, 2017 ; Rafiq et al., 2014 ; Röttinger et al., 2004 ). During skeletogenesis, MAPK phosphorylates Ets1, leading to activation and increased transcription of Alx1, followed by expression and activation of skeletogenic products. For echinoderm skeletogenesis to occur, three elements are necessary: 1) A source of MAPK/ERK signal activation, 2) Ets1 expression and phosphorylation, 3) Alx1 expression. Ancestrally, Ets1 likely specified both adult skeleton and larval mesoderm in echinoderms (Koga et al., 2010 ). Our results from this study confirm previous work on MAPK activation via THs in sea urchins (Taylor and Heyland 2018 ), and provide new evidence for this mechanism in sea stars, and brittle stars. These findings support the hypothesis that an integrin receptor binding THs and capable of MAPK signal transduction is a common feature of echinoderm skeletogenic mesenchyme. Recent work further supports this hypothesis, as T4 and to a lesser extent T3 bind to membrane fractions of sea urchin embryos (Taylor et al. 2023 ). A putative RGD-binding integrin which is a likely candidate (Integrin αPβG) has been reported to have high expression in sea urchin gastrula PMCs, the skeletogenic cells during early sea urchin development (Cocurullo et al. 2023 , Susan et al. 2000, Marsden and Burke 1997). Expression of RGD-binding proteins is crucial for the epithelial-mesenchyme transition in echinoderms (Hertzler & McClay, 1999 ; Katow, 2015 ), vertebrates (Eliceiri & Cheresh, 1998 ; Ludwig et al., 2021 ; Nieberler et al., 2017 ), and non-bilaterians (Magie & Martindale, 2008 ), and may predate multicellular life (Custodio et al., 1995 ). RGD-binding of integrins to an extracellular matrix allows for selective adhesion, detection of the ECM, and signal transduction, which may have contributed to the evolution of multicellularity. Sponges and corals–some of the earliest branching metazoans–utilize iodinated tyrosine residues in the construction of the skeletal matrix, which in some sponges and corals is composed of up to 10–26% iodine (dry weight), predominantly in the form of iodinated scleroproteins (Roche 1952 , Goldberg 1978). The greatest fraction of the iodinated tyrosine is in the form of T2 and T4 (Roche 1952 , Nowak et al. 2009 ) with exposure to T4 increasing the skeletal deposition rate in corals (Kingsley et al., 2001 ; Nowak et al., 2009 ). It is therefore conceivable that thyroid hormones served as a structural element of early metazoan ECM. It is not clear whether T4 in corals has a regulatory effect, or if increased skeletal deposition rate as a result of T4 exposure is a consequence of increased material availability. In contrast, the primary active form of THs in vertebrates, T3, has not been detected in non-bilaterians (Tarrant, 2005, Roche 1952 ). We speculate that the capability of RGD-binding integrins to bind T4 and other iodinated tyrosine compounds may have an ancient origin in non-bilaterians (discussed in Taylor & Heyland, 2017 ), though the mechanism of action and function of T4 in non-bilaterians remains unknown. Echinoderms possess a single THR gene, orthologous to THRβ, and while binding of THs to the echinoderm THR has not been demonstrated, we have previously shown that T4 can regulate gene expression of genes proximal to TH response elements in the genome (Taylor et al. 2023 ). Furthermore, T4 can bind to nuclear extract from echinoid cells (Saito et al. 2012). Our results here show TH binding to the nucleus in sea star and sea urchin gut and rudiment. We only observed this binding in late-stage larvae, corroborating our previous hypothesis that T4 regulation of gene expression via THR is a late-larval phenomenon in sea urchins (Taylor et al. 2023 ). In this study, T4 exposure resulted in an increase of TH binding locations, an effect which was not inhibited by RGD peptide in sea urchin, ophiuroids and sea star larvae. Autoregulation of the THR is a classic sign of canonical TH signaling via a nuclear receptor, an effect which we also detected in Taylor et al. ( 2023 ). Since RGD is not known to bind to the nuclear hormone receptor, this suggests that expression of TH binding locations may be under control of a non-integrin mechanism, such as via autoregulation by the THR, providing evidence for canonical TH signaling activity in all three classes of Echinoderm we examined. The commonality of binding locations and the evidence that THs may regulate metamorphic development in echinoids (Chino et al., 1994a ; Heyland et al., 2004 ; Saito et al., 1998 ) and sea stars (Johnson and Cartright 1996) suggests a potential role for THR in regulating echinoderm metamorphosis. However, unpublished data mentioned in Holzer et al. (2017) suggests that T4, among other TH compounds, may not bind to sea star isolated THR, and these authors hypothesized that THR may be functioning while unliganded. Additionally, it appears that retinoic acid signaling via the retinoic acid receptor (RAR) instead may be a mechanism for nuclear hormone receptor-regulated metamorphosis of sea stars, as in crinoids (Yamakawa et al., 2018 , 2020 ). Evidence for genomic regulation of development by THs by binding to a THR is strong in sea urchins and tenuous for sea stars and brittle stars. Future research will have to test this hypothesis further by analyzing transcriptomic response to THs (as in Taylor et al. 2023 ), ideally with a ChIP assay or binding kinetics to confirm TH binding to THR. Thyroid hormone signaling has been repeatedly co-opted to regulate developmental processes. Not only do THs work via multiple independently evolved mechanisms (Davis et al., 2016 ), THs regulate developmental processes which also evolved independently. For example, THs appear to regulate skeletogenesis in both echinoderms and chordates – but echinoderm skeletogenesis is a novelty of the phylum and distinct in both regulatory mechanisms and material components from chordate skeletogenesis (Ben-Tabou de-Leon, 2022 ; Livingston et al., 2006 ; Murdock, 2020 ; Rafiq et al., 2014 ). THs additionally regulate metamorphic development or settlement in molluscs, annelids, echinoderms, and chordates (Carpizo-Ituarte & Rosa-Velez, 1993 ; Chino et al., 1994b ; Fukazawa et al., 2001 ; Heyland et al., 2004 ; Holzer, 2015 ; Huang et al., 2015 ; Johnson, 1998 ; Klootwijk et al., 2011 ; Paris et al., 2008 , 2010 ; Saito et al., 1998 ), a process which may or may not share a single origin. As well, THs regulate diverse other developmental systems, including neurogenesis, vasculogenesis, metabolism, and myogenesis (Brent, 2012 ; Davis et al., 2016 ). THR actions rely on RXR, a transcription factor which is already implicated in a large number of transcriptional regulatory events (Evans & Mangelsdorf, 2014 ) and creates a signaling complex with THR. The genomic TH mechanism has the ability to activate and regulate a long list of genes (Taylor et al., 2023 ; Wang et al., 2023 ) and RXR is ubiquitously expressed (Evans & Mangelsdorf, 2014 ; Viera-Vera & García-Arrarás, 2018 ), meaning coexpression of THR and TH availability are the main regulators of cell responsiveness to THs via the genomic pathway. Synthesis and metabolism of THs is common to most metazoans, as most classes, with the notable exception of insects, have the ability to synthesize thyroxine and other THs (Taylor & Heyland, 2017 ). Nuclear hormone receptor heterodimers with RXR as a component are implicated in control of development and metamorphosis in metazoans (Hall & Thummel, 1998 ; Laudet, 2011 ), including cnidarians which do not possess a THR (Fuchs et al., 2014 ). These features combined create a readily evolvable system of TH gene regulation. Similarly, non-genomic signaling acts via integrin activation of MAPK, a ubiquitous pathway which can be triggered by a commonly expressed class of proteins: RGD-binding integrins (Bergh et al., 2005 ; Cody et al., 2007 ; Davis et al., 2016 ; Taylor et al., 2023 ; Taylor & Heyland, 2018 ). Receptiveness to integrin mediated signaling can easily evolve in multiple cell types, relying only on proximity to a source of THs and expression of a TH-binding integrin making TH signaling a versatile system in both development and evolution. In a cell containing both signaling pathways, we speculate that external THs would first activate the integrin signaling pathway, triggering rapid phosphorylation and activation of proteins (including the nuclear THR) before being transported to the cytoplasm/nucleus and binding to the nuclear THR to regulate gene transcription. Thyroid hormone regulation of skeletogenic mesenchyme appears to be a common mechanism in echinoderms, including conserved expression of an integrin receptive to T4 which can trigger a MAPK cascade, and a conserved requirement for a MAPK cascade phosphorylating Ets1 and initiating skeletogenesis via Alx1 (Czarkwiani et al., 2013 ; Gao & Davidson, 2008 ; Koga et al., 2016 ; McCauley et al., 2012 ; Röttinger et al., 2004 ). Skeletogenesis is an essential component of metamorphic development and acceleration of skeletogenesis may play a role in regulation of metamorphosis, particularly in echinoid larvae (Heyland & Hodin, 2014 ; Parks et al., 1988 ; Taylor & Heyland, 2018 ; Wray & Raff, 1990 ). TH levels in sea urchins rise prior to metamorphosis (Chino et al., 1994), contributing to the control of developmental timing of metamorphosis and settlement. The acceleration of skeletogenesis by THs represents an extremely evolvable mechanism, as THs would already be present in and proximal to the rudiment, and skeletogenic mesenchyme would already be sensitive to MAPK phosphorylation. MAPK regulation of skeletogenic activity seems to be present in all echinoderms. The only necessary element would be expression of a TH-binding integrin to trigger or enhance MAPK signaling. TH regulation of skeletogenesis appears to exist in sea urchins, sea stars, and brittle stars. Alternatively, the inverse could apply: TH regulation of metamorphosis would be extremely evolvable in the case that THs are already synthesized or transported to the rudiment to control skeletogenesis. THs accelerate both larval and adult skeletogenesis in larval brittle stars and sea urchins (and adult skeletogenesis in sea star larvae). It seems increasingly likely that TH regulation of echinoderm skeletogenesis is at least as ancient as the divergence of Echinozoa and Asterozoa. Critically, the hypothesis that endogenous hormone synthesis is responsible for regulation of skeletogenesis in larval and adult echinoderms is still mostly untested. TH mechanisms in echinoid metamorphic development may involve peroxidase-facilitated diffusion or transport (Heyland et al., 2006 ; Miller & Heyland, 2013 ), but the ultimate source of thyroid hormones in embryonic skeletogenesis remains unclear. Additionally, responsiveness to THs in echinoderm gastrulae may be a consequence of a shared GRN in gastrulae, larvae, and adults, and not a trait which is active under normal physiological conditions. Future work should focus on inhibition of TH synthesis and attempt to determine which effects of THs, if any, are derived from endogenous hormone synthesis versus exogenous hormone sources (as in the vitamone hypothesis; Eales, 1997 ), as well as the source of thyroid hormones in Echinoderm embryos and larvae. 4.2 Skeletal loss/reduction in sea stars and sea cucumbers In the case of a single larval origin of skeletogenesis, there is currently no satisfactory explanation for the loss of skeleton in sea star larvae. Sea cucumbers and sea stars have likely undergone an independent loss of larval skeleton in comparison to sea urchins and brittle stars. The reduction in sea cucumber larval skeleton might be attributed to the same evolutionary process as the reduction in adult skeleton, given that they share a GRN (McCauley et al., 2012 ; Zhang et al., 2017 ), and expression of biomineralization-related genes is greatly reduced in the adult (A. B. Smith & Reich, 2013 ; Zhang et al., 2017 ). It is possible that the adult skeleton of sea stars has also been reduced, as they possess less skeleton (dry weight) than either sea urchins or brittle stars (Dubois, 2014 ). The adult skeleton is highly reduced in sea cucumbers, some crinoids, and some extinct echinoderms (Zamora et al. 2022, Smith and Reich 2013 , Smirnov 2017). In contrast with sea star larvae, sea cucumbers still initiate larval skeletogenesis and have larval skeletogenic mesenchyme. It is likely that the sea cucumber larval skeleton was reduced from an ancestral state, given the close relation to sea urchins and brittle stars, both of which have extensive larval skeletons. It has been suggested that sea cucumber adult skeletal reduction is an example of paedomorphosis (Cuénot, 1948; Smirnov, 2015); however, it is not clear whether reduction in larval or adult skeletogenesis came first, or if due to the shared regulatory mechanism, the reduction was simultaneous. The secondary reduction in sea cucumber skeleton appears to be a likely result of modification or loss of transcription factors in the skeletogenic mesenchyme, possibly related to the reduction in adult skeleton. This contrasts with reduced sea star skeleton which relies on non-expression of the skeletogenic transcription factors until metamorphic development. For this reason, we hypothesize that sea cucumber larvae may still be responsive to thyroid hormone exposure and predict that they might undergo accelerated or expanded skeletogenesis after TH exposure. This could be tested in future experiments and would shed light on both sea star and sea cucumber skeletogenesis, as well as the differing evolutionary trajectories that led to reduction/loss of larval skeletogenesis in both lineages. Sea star larvae do not express Alx1 and are therefore incapable of producing skeleton until metamorphic development when coelom-derived mesenchyme likely becomes primed for skeletogenesis by expressing Alx1 and other skeletogenic regulatory genes (Koga et al., 2016 ). In contrast, sea cucumbers retain expression of Alx1, but have lost expression of downstream secondary transcription factors in the skeletal mesenchyme (McCauley et al., 2012 ). This explains why sea star gastrulae were unresponsive to THs in our experiments: Alx1 is not present to be phosphorylated and therefore no skeleton can be formed (Koga et al., 2016 ). The full complement of skeletogenic regulator genes is present and functional in sea stars, but not expressed in larval cells until the production of skeleton in the rudiment prior to metamorphosis. This may represent a secondary loss of the previously evolved heterochronic activation of the skeletogenesis program during larval gastrulation (Shashikant et al., 2018 ). 4.3. Thyroid hormone regulation of developmental timing Regulation of developmental timing is critically important in the case of both larval and juvenile skeletogenesis. Larval skeleton in sea urchins and brittle stars supports the larval arms which act as both feeding structures and a defence against predation (Boidron-Metairon, 1988 ; Strathmann et al., 1992 ). Skeletogenesis must begin soon after hatching, as the larvae prepare to feed. In late-stage larvae, development to metamorphosis and juvenile skeletogenesis are essential steps to prepare for life on the benthos. P. ochraceus can produce skeleton weeks before metamorphosis (Pia et al., 2012 ). S. purpuratus , develops skeletal elements 5–7 days prior to metamorphosis, and O. aculeata produces skeleton only one or two days before metamorphosis. These represent differing strategies of resource investment in juvenile structures during the larval feeding stage. Control of resource allocation towards metamorphic development must be carefully balanced with larval growth (Strathmann et al., 1992 ). For instance, in sea urchins, THs increase skeletogenesis in the juvenile rudiment while shortening the larval arms (Armstrong & Lessios, 2015 ; Chino et al., 1994a ; Heyland et al., 2004 ; Wynen et al., 2022 ). Similarly, THs may allow for control of resource investment in both brittle star and sea star larvae. One potential source of THs is exogenous (Eales, 1997 ; Heyland & Moroz, 2005 ), implying that food availability might modulate late larval development. P. ochraceus is also notable for spending a longer than typical time developing juvenile structures as a brachiolaria larva (Pia et al., 2012 ). The early responsiveness of P. ochraceus skeletogenesis to THs may relate to the need for temporal control of the extended development to metamorphosis. In contrast, O. aculeata develops few skeletal structures prior to a rapid metamorphosis and was only responsive to TH acceleration of juvenile skeletogenesis several days before a typical metamorphosis would occur. We did not examine any direct-developing echinoderms (echinoderms which develop directly from egg to adult without a feeding larval stage). Echinoderm eggs are maternally provisioned with THs (Chino et al., 1994a ) and TH regulation of metamorphic development is sufficient to allow an obligate feeding species with an indirect development to act as a facultative feeding larva and metamorphose without the need for food (Heyland et al. 2004 ), albeit with a much smaller juvenile size. Based on the potentially shared mechanisms and role of THs in sea stars and brittle stars, we predict that the same phenomenon might be observed in non-echinoid echinoderms. 5. Conclusions Thyroid hormones, principally thyroxine, accelerate skeletogenesis in sea urchins, sea stars, and brittle star larvae. THs also accelerate skeletogenesis in brittle star and sea urchin gastrulae, but do not induce ectopic skeletogenesis in sea star gastrulae (which normally do not produce skeleton). Thyroid hormones bind to cells proximal to regions of skeletogenesis, primarily in the gut and rudiment, and stimulate MAPK phosphorylation. RGD peptide, an inhibitor of the RGD binding pocket in RGD-binding integrins, inhibits the effect of thyroid hormones in all three echinoderm classes examined. PD98059, an inhibitor of MAPK signaling, prevents the effect of THs on skeletogenesis, especially in sea star larvae. Thyroid hormones may act non-genomically, via a membrane integrin receptor-mediated MAPK cascade in sea stars, brittle stars, and sea urchins. TH regulation of mesenchyme cell activity may be an ancient mechanism to control timing of development, including skeletogenesis. TH regulation of skeletogenesis in late mesenchyme cells prior to metamorphosis may have been co-opted to regulate larval skeletogenesis in sea urchins and brittle stars. Declarations Author Contribution All authors contributed to the conception, design, interpretation, and revisions of this manuscript. E.T. wrote the manuscript draft, conducted the experiments, and analyzed the data. References Armstrong, A. F., & Lessios, H. A. (2015). The evolution of larval developmental mode: Insights from hybrids between species with obligately and facultatively planktotrophic larvae. Evolution & Development , 17 (5), 278–288. https://doi.org/10.1111/ede.12133 Ben-Tabou de-Leon, S. (2022). 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(A) MAPK phosphorylation was quantified by automatic cell counting in the Hoechst 33342 channel, followed by a measurement of fluorescent intensity of each individual cell in a radius around each nucleus (see file S1. for ImageJ script). (B) Intensity was normalized to the Hoechst 33342 signal to account for attenuation S2.pdf Figure S2. RGD effects on muscle contraction in Pisaster ochraceus. RGD peptide induced repeated whole-body muscle contractions in P. ochraceus bipinnaria. This effect was observed to a lesser degree in brachiolaria. S3.pdf Figure S3. MAPK and T4 binding site distribution in Dendraster excentricus gastrula exposed to T4. Gastrula was exposed to 10 -7 M T4 for 90 minutes. Gastrula was stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). Both T4 binding sites and MAPK activation are ubiquitous during early gastrulation. (A) Maximum intensity projection of confocal fluorescent image of D.e. gastrula, 30 hours post-fertilization. (B) Close-up side view of lateral ectoderm. (C) Close-up of extracellular matrix and basal membrane near ventrolateral cluster (vlc). (D) Nucleus and cytoplasm of cells near ventrolateral cluster. S4.pdf Figure S4. Ectopic skeleton and accelerated skeletogenesis in Dendraster excentricus . (A) Whole-larva view of skeleton. Additional skeleton was observed in the larval arms and posterior end. (B) Ectopic arm skeleton formed duplicate parallel larval arms, connected with a “window-like” network. (C) Ectopic skeleton at the posterior end formed a network reminiscent of late-larval development, similar to a juvenile plate. (D) Larvae were exposed to the thyroid hormones, rT3, T3, and T4 (10 -7 M), RGD peptide (RGD; 10 -7 M) or PD98059 (PD; 5x10 -5 M). Thyroid hormones, including T4 and T3, accelerate skeletogenesis in. RGD peptide and PD98059 inhibit the effects of THs on skeletogenesis (t-test, p < 0.05). Cite Share Download PDF Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Developmental Biology Advances → Version 1 posted Editorial decision: Revision requested 23 Feb, 2024 Reviews received at journal 12 Feb, 2024 Reviewers agreed at journal 03 Feb, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers invited by journal 27 Jan, 2024 Editor assigned by journal 16 Jan, 2024 Submission checks completed at journal 16 Jan, 2024 First submitted to journal 12 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3858209","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267613968,"identity":"253039f8-2934-45a5-930e-ba2bcba6b59f","order_by":0,"name":"Elias Taylor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACdsYGBoYKMJPxAIhNGDCDlJ2BsInVAjK+jRQt/MzMjR9/zjssJx+RfOBw4Q4GeX5C2iSbGZulebcdNja8kZZweOYZBsMZBwhoMTjM2CDNuO1w4sYZOQaHedsYEhgIabE/zNj88+cckJb8D2At8gRtYWZsk+BtOJw4XyKHAazFgJAWicOMbdY8x9KNDXieGRye2SZhuJGQFv729sc3f9RYy8m3Jz98XNhmIy9HSAvChQfAcSRBrHogkG+AROsoGAWjYBSMAgwAABwwQxPAjk63AAAAAElFTkSuQmCC","orcid":"","institution":"University of Guelph","correspondingAuthor":true,"prefix":"","firstName":"Elias","middleName":"","lastName":"Taylor","suffix":""},{"id":267613969,"identity":"79f46dc0-2fd0-449e-8707-18784c8c1b90","order_by":1,"name":"Andreas Heyland","email":"","orcid":"","institution":"University of Guelph","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Heyland","suffix":""}],"badges":[],"createdAt":"2024-01-12 21:15:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3858209/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3858209/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13227-024-00226-2","type":"published","date":"2024-08-07T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49792831,"identity":"f890f82b-b839-4962-85a9-e5905befee27","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192307,"visible":true,"origin":"","legend":"\u003cp\u003eHypotheses about the evolution of skeletogenesis across echinoderm taxa with a possible ancestral function of non-genomic thyroid hormone signaling. Recent evidence suggests a single origin of larval skeletogenesis followed by loss in Asteroids (sea stars; Erkenbrack \u0026amp; Thompson, 2019), although independent origins of larval skeletogenesis in Ophiuroids (brittle stars) and Echinoids/Holothuroids (sea urchins/sea cucumbers) have also been proposed (Cary \u0026amp; Hinman, 2017; Dylus et al., 2016; McCauley et al., 2012). We test the hypothesis that THs, predominantly T4, regulate skeletogenesis in Ophiuroids, Echinoids, and Asteroids, as a result of evolutionarily ancient regulation of skeletogenesis by THs. We suggest that TH regulation was co-opted to regulate larval skeletogenesis when the gene regulatory apparatus allowing for skeletogenesis in adults became expressed and co-opted by the larval stage of some echinoderms.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/4533deede84b2975065b20f4.png"},{"id":49793083,"identity":"d9c759aa-37bb-4609-a574-8f49a9174af8","added_by":"auto","created_at":"2024-01-18 06:13:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1467685,"visible":true,"origin":"","legend":"\u003cp\u003eLarval and juvenile skeletogenesis in sea star, sea urchin, and brittle star larvae. Larval skeleton is depicted in red, while juvenile skeleton is depicted in green. (\u003cstrong\u003eA\u003c/strong\u003e) Sea star gastrula (\u003cem\u003ePisaster ochraceus\u003c/em\u003e). No skeleton is present. (\u003cstrong\u003eB\u003c/strong\u003e) Sea star brachiolaria (\u003cem\u003eP. o.\u003c/em\u003e). Adult skeleton develops in the somatocoel/rudiment. (\u003cstrong\u003eC\u003c/strong\u003e) Close-up of sea star rudiment (\u003cem\u003eP. o\u003c/em\u003e.) skeletogenesis begins with small spicules, developing into the plates of the juvenile test. (\u003cstrong\u003eD\u003c/strong\u003e) Sea urchin gastrula (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e). Skeletogenesis begins with spicules at the ventrolateral clusters. These spicules will elongate and form the post-oral and antero-lateral larval arms. (\u003cstrong\u003eE\u003c/strong\u003e) Sea urchin pluteus larva (\u003cem\u003eS. p.\u003c/em\u003e). juvenile skeletogenesis typically begins with the genital plate adjacent to the gut and tube feet/juvenile spines in the rudiment. (\u003cstrong\u003eF\u003c/strong\u003e) Close-up of sea urchin rudiment (\u003cem\u003eS. p.\u003c/em\u003e) Tube feet and spines in the rudiment are among the first skeletal structures formed. (\u003cstrong\u003eG\u003c/strong\u003e) Brittle star gastrula (\u003cem\u003eOphiopholis aculeata\u003c/em\u003e) Skeletogenesis begins with spicules at the ventrolateral clusters. These spicules will elongate and form the post-oral and anterolateral larval arms. (\u003cstrong\u003eH\u003c/strong\u003e) Brittle star ophiopluteus (\u003cem\u003eO. a.\u003c/em\u003e) Adult skeletogenesis begins adjacent to the gut with the formation of terminal and radial plates. (\u003cstrong\u003eI\u003c/strong\u003e) Close-up of ophiopluteus (\u003cem\u003eO. a.\u003c/em\u003e) gut and hydrocoel. \u003cstrong\u003ear\u003c/strong\u003e: archenteron, \u003cstrong\u003ehc\u003c/strong\u003e: hydrocoel, \u003cstrong\u003esc\u003c/strong\u003e: somatocoel, \u003cstrong\u003erud\u003c/strong\u003e: rudiment, \u003cstrong\u003emg\u003c/strong\u003e: midgut, \u003cstrong\u003eop\u003c/strong\u003e: oral plate, \u003cstrong\u003emp\u003c/strong\u003e: madreporitic plate, \u003cstrong\u003ela\u003c/strong\u003e: larval arm, \u003cstrong\u003ejs\u003c/strong\u003e: juvenile spine, \u003cstrong\u003egp\u003c/strong\u003e: genital plate, \u003cstrong\u003etf\u003c/strong\u003e: tube foot, \u003cstrong\u003etp\u003c/strong\u003e: terminal plate, \u003cstrong\u003erp\u003c/strong\u003e: radial plate.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/dbf076ea0842546150bcf5ee.png"},{"id":49792844,"identity":"f28cd40c-162a-4e10-8a2a-ccfdaf26e294","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":406959,"visible":true,"origin":"","legend":"\u003cp\u003eThyroid hormones accelerate skeletogenesis in sea urchins, sea stars, and brittle stars. Triangles indicate skeletogenesis. (A) Sea urchin gastrula in control conditions. (B) Sea urchin gastrula after T4 exposure for 20 hours at 10\u003csup\u003e-7\u003c/sup\u003e M. Skeletogenesis is taking place in the ventrolateral clusters. (C) Sea urchin pluteus gut and rudiment. Skeletogenesis is visible in the genital plate and as a small single spicule in the rudiment. (D) Sea urchin pluteus gut and rudiment after T4 exposure for 5 days at 10\u003csup\u003e-7\u003c/sup\u003e M. Skeletogenesis is dramatically accelerated with multiple skeletal plates, juvenile spines, and tube foot rings observed. (E) Ophiuroid gastrula at the beginning of skeletogenesis. (F) Ophiuroid gastrula after T4 exposure for 24 hours at 10\u003csup\u003e-7\u003c/sup\u003e M. Skeletogenesis has been accelerated and the larval arms have begun to form. (G) Ophiuroid ophiopluteus gut region. Somatocoel has not yet begun to form skeleton. (H) Ophiuroid ophiopluteus gut region after T4 exposure for 48 hours at 10\u003csup\u003e-7\u003c/sup\u003e M. Several skeleton spicules are visible. (I) Sea star early bipinnaria. No skeletogenesis is present. (J) Sea star early bipinnaria after T4 exposure during gastrulation for 3 days at 10\u003csup\u003e-7\u003c/sup\u003e M. No skeletogenesis is present. (K) Sea star late bipinnaria gut. No skeletogenesis is present. (L) Sea star late bipinnaria gut after T4 exposure for 24 hours at 10\u003csup\u003e-7\u003c/sup\u003e M. T4 dramatically accelerates skeletogenesis adjacent to the gut. (M) Quantification of skeletogenesis during gastrulation reveals that thyroxine (T4) accelerates skeletogenesis in sea urchin and ophiuroid gastrulae, but not sea star larvae (n = 24-40, t-test, p \u0026lt; 0.05). (N) Quantification of juvenile skeletogenesis reveals that thyroxine (T4) accelerates skeletogenesis in sea urchin, ophiuroid, and sea star late-stage larvae (n = 12-30, t-test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/c7b14f4358a8d738a1928f93.png"},{"id":49793085,"identity":"0070dc90-c048-401d-b0bf-1ac6fe8940a3","added_by":"auto","created_at":"2024-01-18 06:13:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210392,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitors of MAPK phosphorylation and integrin binding inhibit the effect of thyroid hormones on skeletogenesis in sea urchin, brittle star, and sea star gastrulae and larvae. L: Larval skeletogenesis, J: Juvenile skeletogenesis. Larvae were exposed to the thyroid hormones, rT3, T3, and T4 (10\u003csup\u003e-7\u003c/sup\u003e M), RGD peptide (RGD; 10\u003csup\u003e-7\u003c/sup\u003e M) or PD98059 (PD; 5x10\u003csup\u003e-5\u003c/sup\u003e M). \u003cstrong\u003e(A)\u003c/strong\u003e Sea urchin gastrulae. Thyroid hormones, including T4 and T3, accelerate skeletogenesis in sea urchin gastrulae (n = 20, F(7,152) = 9.20; Tukey’s HSD, p \u0026lt; 0.00001, T4: 95%CI[2.06-fold, 4.16-fold], T3: 95%CI[1.57-fold, 2.62-fold]). RGD peptide and PD98059 inhibit the effects of THs on skeletogenesis (Tukey’s HSD, p \u0026lt; 0.00001). Data reproduced from (Taylor et al. 2023). \u003cstrong\u003e(B)\u003c/strong\u003e Sea star gastrulae. THs were not found to induce skeletogenesis in sea star gastrulae (n = 20, ANOVA not possible, all groups identical; p = 1.00). Sea star gastrulae do not contain the cell type necessary for skeletogenesis. \u003cstrong\u003e(C)\u003c/strong\u003e Brittle star gastrulae. Thyroid hormones, including T4 and T3, accelerate skeletogenesis in Brittle star gastrulae (n = 20, F(7,152) = 12.45; p \u0026lt; 0.00001; Tukey’s HSD, T4: 95%CI[1.75-fold, 2.69-fold], T3: 95%CI[1.08-fold, 2.03-fold]). RGD peptide (RGD) and PD98059 (PD) partially inhibit the effects of THs on skeletogenesis (Tukey’s HSD, p \u0026lt; 0.0001). \u003cstrong\u003e(D) \u003c/strong\u003eSea urchin pluteus larva. Thyroid hormones accelerate juvenile skeletogenesis (n = 12, F(10,121) = 20.88, p \u0026lt; 0.00001), even in the presence of RGD peptide, or PD98059 (p = 0.0023, 0.019). A higher dosage of RGD peptide/PD98059 was previously found to inhibit skeletogenesis (Taylor 2021).\u003cstrong\u003e \u003c/strong\u003eData reproduced from (Taylor et al. 2023). \u003cstrong\u003e(E) \u003c/strong\u003eSea star bipinnaria larva.\u003cstrong\u003e \u003c/strong\u003eThyroid hormones accelerate, including T4 and T3, juvenile skeletogenesis (n = 6, F(7,40) = 36.25, p \u0026lt; 0.00001). The effect of thyroid hormones is inhibited by PD9809 and RGD peptide (Tukey’s HSD, p \u0026lt; 0.00001). \u003cstrong\u003e(F) \u003c/strong\u003eOphiopluteus larva. Thyroid hormones (T4) accelerate juvenile skeletogenesis (n = 12, F(3,44) = 24.22; Tukey’s HSD p \u0026lt; 0.00001). This effect is partially inhibited by PD98059 (p = 0.0163). The effect of RGD peptide on skeletogenesis was not tested in late-stage \u003cem\u003eO. aculeata\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/cb7e8115a1ad7fe23e168720.png"},{"id":49792836,"identity":"c2860188-dc7d-480f-93ce-bcf62a360461","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3119128,"visible":true,"origin":"","legend":"\u003cp\u003ePutative MAPK-P and T4 binding site distribution in \u003cem\u003eStrongylocentrotus purpuratus \u003c/em\u003egastrulae. Gastrulae were stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). Fluorescent debris exterior to gastrula was removed. \u003cstrong\u003e(A)\u003c/strong\u003e T4 increases MAPK intensity in S. p. larvae (n = 4, p \u0026lt; 0.05). This effect may be partially inhibited by RGD peptide (p \u0026gt; 0.05). \u003cstrong\u003e(B) \u003c/strong\u003eMaximum intensity projection of confocal fluorescent image of \u003cem\u003eS. p.\u003c/em\u003e gastrula. (\u003cstrong\u003eC-C‴\u003c/strong\u003e) Focal slice of gastrula showing primary mesenchyme cells (PMCs) and lower archenteron. Triangles indicate PMCs. PMCs display greater T4 binding and MAPK phosphorylation than surrounding cells. Ventrolateral clusters have formed (vlc). (\u003cstrong\u003eD-D‴\u003c/strong\u003e) Focal slice of gastrula showing tip of the archenteron and developing coelomic pouches, as well as secondary mesenchyme cells. Triangles indicate SMCs. SMCs and the coelomic pouches display greater MAPK phosphorylation than surrounding cells. T4 binding is slightly elevated in regions near the tip of the archenteron and small protein clusters in SMCs. \u003cstrong\u003ec\u003c/strong\u003e: Coelomic pouch, \u003cstrong\u003ear\u003c/strong\u003e: Archenteron (gut), \u003cstrong\u003epmc\u003c/strong\u003e: primary mesenchyme cells, \u003cstrong\u003esmc\u003c/strong\u003e: secondary mesenchyme cells, \u003cstrong\u003evlc\u003c/strong\u003e: ventrolateral cluster.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/fef6f4d8e5e5237beb9933fd.png"},{"id":49792832,"identity":"7f66fea2-c0c6-4cb3-bd81-e938a4dd017b","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2390203,"visible":true,"origin":"","legend":"\u003cp\u003eMAPK-P and T4 binding site distribution in \u003cem\u003ePisaster ochraceus \u003c/em\u003egastrulae. Gastrulae were stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). \u003cstrong\u003e(A) \u003c/strong\u003eMaximum intensity projection of confocal fluorescent image of control P.o. gastrula. \u003cstrong\u003e(B)\u003c/strong\u003e Maximum intensity projection of confocal fluorescent image of gastrula exposed to T4 for 90 minutes. (\u003cstrong\u003eC-C’\u003c/strong\u003e) Focal slice of control gastrula showing tip of the archenteron and developing coelomic pouches. (\u003cstrong\u003eD-D’\u003c/strong\u003e) Focal slice of T4-exposed gastrula showing tip of the archenteron and developing coelomic pouches. MAPK phosphorylation patterns are similar to the control gastrula. Few to no T4-binding sites were observed in any early \u003cem\u003eP. ochraceus \u003c/em\u003egastrulae. \u003cstrong\u003e(E)\u003c/strong\u003e T4 did not significantly increase MAPK phosphorylation in P.o. gastrulae. RGD peptide decreased MAPK phosphorylation (n = 5, p \u0026lt; 0.05). \u003cstrong\u003ec\u003c/strong\u003e: Coelomic pouch, \u003cstrong\u003ear\u003c/strong\u003e: Archenteron (gut)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/a8c9266bbae00be2445358a6.png"},{"id":49793087,"identity":"a0123cfe-4633-41c9-ade8-3d6089d72fcc","added_by":"auto","created_at":"2024-01-18 06:13:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1258168,"visible":true,"origin":"","legend":"\u003cp\u003eMAPK and T4 binding site distribution in \u003cem\u003ePisaster ochraceus \u003c/em\u003elarva (bipinnaria). Bipinnaria was stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). \u003cstrong\u003e(A) \u003c/strong\u003eMaximum intensity projection of confocal fluorescent image of \u003cem\u003eP.o.\u003c/em\u003e bipinnaria. \u003cstrong\u003e(B-B″’)\u003c/strong\u003eFocal slice of midgut wall. \u003cstrong\u003e(C-C″’)\u003c/strong\u003e Focal slice showing ventral view of midgut wall.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/5595071882dc4ccf724307d7.png"},{"id":49792843,"identity":"6cadf86b-e0bd-487c-b379-3941ce8fc0b5","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4515982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Maximum intensity projection of a \u003cem\u003ePisaster ochraceus\u003c/em\u003e larva (brachiolaria) which has just begun rudiment development. \u003cstrong\u003e(B)\u003c/strong\u003e T4 increased MAPK phosphorylation in P.o. late bipinnaria/early brachiolaria (n = 6, p \u0026lt; 0.05). RGD peptide decreased MAPK phosphorylation (n = 6, p \u0026lt; 0.05). \u003cstrong\u003e(C)\u003c/strong\u003eSingle slice of MAPK phosphorylation in midgut. \u003cstrong\u003e(C′)\u003c/strong\u003e Single slice of T4-binding locations in midgut. \u003cstrong\u003e(D)\u003c/strong\u003e Coelom adjacent to gut. \u003cstrong\u003e(D′)\u003c/strong\u003eMAPK phosphorylation in gut and coelom. \u003cstrong\u003e(D″)\u003c/strong\u003e T4 binding locations in gut. Coelom shows little T4-binding. \u003cstrong\u003e(E)\u003c/strong\u003e Nuclear stain of hindgut. \u003cstrong\u003e(E′)\u003c/strong\u003eMAPK phosphorylation in hindgut. \u003cstrong\u003e(E″)\u003c/strong\u003e T4 binding locations in hindgut. \u003cstrong\u003ehc\u003c/strong\u003e: hydrocoel, \u003cstrong\u003egut\u003c/strong\u003e: midgut, \u003cstrong\u003ei\u003c/strong\u003e: intestine leading to anus, \u003cstrong\u003esc\u003c/strong\u003e: somatocoel.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/24cb77014885ad70556c0201.png"},{"id":49792841,"identity":"5f6a343e-6e64-41d6-aa00-ada887f184e0","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2536190,"visible":true,"origin":"","legend":"\u003cp\u003eThyroid hormones bind and increase MAPK phosphorylation in the somatocoel and in presumptive skeletogenic mesenchyme in the larval arms. \u003cstrong\u003e(A-B)\u003c/strong\u003e Maximum intensity projection of an \u003cem\u003eOphiopholis aculeata \u003c/em\u003elarva (ophiopluteus) after T4 exposure. \u003cstrong\u003e(C)\u003c/strong\u003e T4 increases MAPK intensity in O. a. larvae (n = 4, p \u0026lt; 0.05). This effect may be partially inhibited by RGD peptide (n = 4, p \u0026gt; 0.05). \u003cstrong\u003e(D-D″’) \u003c/strong\u003eSingle focal slice revealing T4 binding locations and MAPK phosphorylation in a larval arm. Triangles indicate presumptive skeletogenic mesenchyme. \u003cstrong\u003e(E-E″’) \u003c/strong\u003eSingle focal slice showing 5-lobed hydrocoel. Triangles indicate hydrocoel lobes. Little to no T4 binding is detected in the hydrocoel and hydrocoel phosphorylation does not differ from control larvae. \u003cstrong\u003e(F-F″’)\u003c/strong\u003eSingle focal slice showing somatocoel adjacent to the gut. Regions of increased T4 binding and MAPK phosphorylation are indicated with triangles. \u003cstrong\u003e(G-G″’) \u003c/strong\u003eSingle focal slice showing gut wall. T4-binding cells, indicated with triangles, do not present with increased MAPK phosphorylation.\u003cstrong\u003e hc\u003c/strong\u003e: hydrocoel, \u003cstrong\u003egut\u003c/strong\u003e: midgut, \u003cstrong\u003esc\u003c/strong\u003e: somatocoel, \u003cstrong\u003ela\u003c/strong\u003e: larval arm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/7fd8d99243210187cbfeb4d9.png"},{"id":49792838,"identity":"ea68b734-016d-4169-816e-d69223de385a","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3030125,"visible":true,"origin":"","legend":"\u003cp\u003eT4 binds to gut and rudiment in \u003cem\u003eS. purpuratus\u003c/em\u003e larvae. Exposure to T4 increases MAPK phosphorylation in the midgut and rudiment. Larvae were stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). Exposure to T4 and RGD peptide increases binding locations for T4 in the midgut. (\u003cstrong\u003eA) \u003c/strong\u003eControl larva. Demonstrates binding sites for T4 in the gut and rudiment cytoplasm/membrane. Triangle indicates an example of a T4 binding site in gut. Arrow indicates T4 binding in rudiment. \u003cstrong\u003e(A′) \u003c/strong\u003eT4 binding locations in control larva. \u003cstrong\u003e(A′′) \u003c/strong\u003eMAPK phosphorylation locations in control larva. Overall MAPK phosphorylation is low.\u003cstrong\u003e (B) \u003c/strong\u003eT4-exposed larva. \u003cstrong\u003e(B′)\u003c/strong\u003e T4 binding locations in gut and rudiment. Triangle indicates an example of a T4 binding site in gut. Arrow indicates T4 binding in rudiment. \u003cstrong\u003e(B′′)\u003c/strong\u003e MAPK phosphorylation in gut and rudiment. Arrow indicates region of greatest phosphorylation in rudiment.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/a84c899e1d6d55e2f51b7fa3.png"},{"id":49792839,"identity":"aa98742b-60a6-420d-9710-4cf6ce4c8f9b","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2384495,"visible":true,"origin":"","legend":"\u003cp\u003eRGD inhibits the effect of T4 in \u003cem\u003ePisaster ochraceus \u003c/em\u003elarvae. T4 binds to gut and somatocoel in \u003cem\u003eP. o. \u003c/em\u003elarvae. Exposure to T4 increases MAPK phosphorylation in the midgut and somatocoel. Larvae were stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). Exposure to T4 and RGD peptide increases binding locations for T4 in the midgut. Triangles indicate T4 binding or MAPK phosphorylation in the gut wall. Arrows indicate T4 binding or MAPK phosphorylation outside the gut wall. \u003cstrong\u003e(A-A′′) \u003c/strong\u003eControl larva, from left to right: Combined image, T4 binding sites, MAPK phosphorylation. T4 binding locations are visible in and adjacent to the gut. \u003cstrong\u003e(B-B′′) \u003c/strong\u003eT4-exposed larva, from left to right: Combined image, T4 binding sites, MAPK phosphorylation. T4 binding locations are visible in and adjacent to the gut, especially adjacent to the somatocoel. Regions of increased MAPK phosphorylation correspond roughly with T4 binding sites. \u003cstrong\u003e(C-C′′) \u003c/strong\u003eT4-exposed larva, from left to right: Combined image, T4 binding sites, MAPK phosphorylation. T4 binding sites are present in the gut wall, but effects of T4 on MAPK phosphorylation are reduced. \u003cstrong\u003es:\u003c/strong\u003e Somatocoel.\u003cstrong\u003e Gut: \u003c/strong\u003emidgut.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/ccb697e61b034c2fcf8df101.png"},{"id":62298905,"identity":"dbf95677-935d-41a3-9e92-964c1a0fa283","added_by":"auto","created_at":"2024-08-12 16:17:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34724723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/eda8d7a0-86cd-40ea-b6ff-f69e1d8d0a70.pdf"},{"id":49792834,"identity":"c867acec-e399-41da-b155-2797927ce9e4","added_by":"auto","created_at":"2024-01-18 06:05:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":190671,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. MAPK quantification methods. (A) MAPK phosphorylation was quantified by automatic cell counting in the Hoechst 33342 channel, followed by a measurement of fluorescent intensity of each individual cell in a radius around each nucleus (see file S1. for ImageJ script). (B) Intensity was normalized to the Hoechst 33342 signal to account for attenuation\u003c/p\u003e","description":"","filename":"S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/974466fa4a5a5674eeeca41a.pdf"},{"id":49793084,"identity":"fe87b180-ee49-4fbf-97ab-b97a9770bbc2","added_by":"auto","created_at":"2024-01-18 06:13:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":137479,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2. RGD effects on muscle contraction in Pisaster ochraceus. RGD peptide induced repeated whole-body muscle contractions in P. ochraceus bipinnaria. This effect was observed to a lesser degree in brachiolaria.\u003c/p\u003e","description":"","filename":"S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/ba0c3135e4667a5bdc6bd796.pdf"},{"id":49793086,"identity":"db269707-bbb2-46ae-9316-d2411ba2acaa","added_by":"auto","created_at":"2024-01-18 06:13:01","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":319290,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3. MAPK and T4 binding site distribution in \u003cem\u003eDendraster excentricus\u003c/em\u003e gastrula exposed to T4. Gastrula was exposed to 10\u003csup\u003e-7\u003c/sup\u003e M T4 for 90 minutes. Gastrula was stained with Hoechst 33342 (blue; nuclei), rhodamine-conjugated T4 (red; T4 binding sites), and an antibody for phosphorylated MAPK (green, P-MAPK). Both T4 binding sites and MAPK activation are ubiquitous during early gastrulation. \u003cstrong\u003e(A) \u003c/strong\u003eMaximum intensity projection of confocal fluorescent image of D.e. gastrula, 30 hours post-fertilization. \u003cstrong\u003e(B)\u003c/strong\u003e Close-up side view of lateral ectoderm. \u003cstrong\u003e(C)\u003c/strong\u003e Close-up of extracellular matrix and basal membrane near ventrolateral cluster (vlc). \u003cstrong\u003e(D)\u003c/strong\u003e Nucleus and cytoplasm of cells near ventrolateral cluster.\u003c/p\u003e","description":"","filename":"S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/853bbd2ad81006629fbf18ab.pdf"},{"id":49792845,"identity":"ac64318a-94f8-44ac-ae19-821155c226f6","added_by":"auto","created_at":"2024-01-18 06:05:02","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":234778,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S4. Ectopic skeleton and accelerated skeletogenesis in \u003cem\u003eDendraster excentricus\u003c/em\u003e. (A) Whole-larva view of skeleton. Additional skeleton was observed in the larval arms and posterior end. (B) Ectopic arm skeleton formed duplicate parallel larval arms, connected with a “window-like” network. (C) Ectopic skeleton at the posterior end formed a network reminiscent of late-larval development, similar to a juvenile plate. (D) Larvae were exposed to the thyroid hormones, rT3, T3, and T4 (10\u003csup\u003e-7\u003c/sup\u003e M), RGD peptide (RGD; 10\u003csup\u003e-7\u003c/sup\u003e M) or PD98059 (PD; 5x10\u003csup\u003e-5\u003c/sup\u003e M). Thyroid hormones, including T4 and T3, accelerate skeletogenesis in. RGD peptide and PD98059 inhibit the effects of THs on skeletogenesis (t-test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"S4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3858209/v1/f1766fa5c36fc2eb05c61945.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThyroid hormones (THs) regulate metamorphosis, skeletogenesis, and diverse physiological/developmental systems in chordate deuterostomes. Evidence also suggests a role for TH signaling in non-chordate bilaterians (Carpizo-Ituarte, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chino et al., 1994; Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Holzer, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sainath et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Saito et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thyroxine (T4) is the most abundant TH analogue in nature. Many non-bilaterians use THs and other iodinated tyrosine derivatives as structural elements or to scavenge free radicals (reviewed in Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). T4 is the typical first step in TH synthesis (Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Edmiston et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Phatarphekar et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Roche, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1952\u003c/span\u003e; Siuda \u0026amp; DeBernardis, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), a biochemical process predating metazoans (Heyland \u0026amp; Moroz, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Phatarphekar et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, the use of THs as a signaling agent to regulate development and metamorphosis can be considered a derived trait of some metazoans.\u003c/p\u003e \u003cp\u003eL-triiodothyronine (T3), a metabolite of T4 and active hormone in vertebrates has been shown to regulate development and metamorphosis (Brtko, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with T4 acting synergistically via non-genomic mechanisms (Das et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In contrast, T4 has roughly ten-fold greater efficacy than T3 on regulation of development, settlement, and metamorphosis in some non-chordates, including sea urchins and molluscs (Chino et al., 1994; Huang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are three major categories of mechanism by which thyroid hormone signal transduction occurs: 1) Genomic signaling via a nuclear hormone receptor (Brent, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), 2) Extra-nuclear actions of a nuclear hormone receptor (Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and 3) Integrin-mediated MAPK phosphorylation cascade (Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The specific mechanisms governing TH actions in non-chordates remain largely unknown, despite new insights from echinoderms, mollusks, and cnidarians. Two main TH receptors, the nuclear thyroid hormone receptor (THR) and RGD-binding integrins are common to all bilaterians and all metazoans respectively (Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). T4 has been shown to accelerate metamorphic development in echinoderms and molluscs (Carpizo-Ituarte, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chino et al., 1994; Fukazawa et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hodin et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Johnson, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Johnson \u0026amp; Cartwright, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Saito et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Evidence suggests that these mechanisms may rely on the nuclear hormone receptor-mediated pathway (Huang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and that THR may regulate apoptosis and skeletogenesis in sea urchins (Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wynen et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, embryonic and larval sea urchin skeletogenesis may be regulated by the TH integrin-mediated pathway (Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaylor and Heyland (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) suggested that sea urchin skeletogenesis is regulated by thyroid hormones via a non-genomic integrin-mediated MAPK cascade triggered by direct TH binding to skeletogenic mesenchyme cells. Additionally, Wynen et al. (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated a role for THs regulating apoptosis in metamorphic development. However, it is unclear if TH signaling and its involvement in skeletogenesis is limited to \u003cem\u003eS. purpuratus\u003c/em\u003e larvae, or if it can be generalized to other sea urchins and echinoderms.\u003c/p\u003e \u003cp\u003eIndirect-developing echinoderms are characterized by a biphasic life cycle with a planktonic phase followed by settlement to the benthos and metamorphosis. Sea urchin, brittle star, and sea star development to settlement involves the production of juvenile structures in the pre-settlement larva, including skeletal elements (e.g. skeletal plates, spines, test, tube-foot rings in sea urchins). The actions of thyroid hormones on skeletogenesis in sea urchins have been of particular interest, as sea urchin larval skeletogenesis is a thoroughly-studied model system (Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In particular, the gene regulatory network (and transcription factors regulating skeletogenesis have been characterized (e.g. Davidson et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Longabaugh, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mann et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; McIntyre et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rafiq et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe development of adult skeleton in a coelom or rudiment adjacent to the gut appears to be an ancient and conserved component of echinoderm development (Isaeva \u0026amp; Rozhnov, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mooi \u0026amp; David, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and is considered a shared characteristic of all extant echinoderms. In contrast, larval skeletogenesis is likely a synapomorphy of holothuroids (sea cucumbers), echinoids (sea urchins), and ophiuroids (brittle stars) (Fig.\u0026nbsp;1; Erkenbrack \u0026amp; Thompson, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Crinoids (sea lilies) and asteroids (sea stars) do not feature larval skeletogenesis (Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Here we use \u0026ldquo;adult skeletogenesis\u0026rdquo; to refer to the development of skeletal elements slated to be incorporated into the juvenile echinoderm, post-settlement (Fig.\u0026nbsp;2). This skeletal development begins prior to settlement, during metamorphic development when skeleton is deposited by a population of mesenchyme cells with a distinct but similar GRN to the primary mesenchyme cells (PMCs) responsible for larval skeleton (Gao \u0026amp; Davidson, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For instance, sea urchins develop spines, portions of the adult test, and skeletal rings supporting the tube feet (Heyland \u0026amp; Hodin, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Much of this development occurs in the juvenile rudiment, a structure adjacent to the larval gut which contains most tissues of the developing juvenile echinoderm. Similarly, the larval ophiuroid, asteroid, and holothuroid produce extensive skeleton prior to metamorphosis, mainly in the juvenile rudiment. In Crinoids, an outgroup to the other extant echinoderms, the skeletonized stalk develops from a coelom adjacent to the gut, while the calyx (analogous to the echinoid test), begins developing from extra-coelomic spicules.\u003c/p\u003e \u003cp\u003eIn many echinoids and ophiuroids, larval skeleton supports the larval arms, important structures for feeding and defense. During embryonic development, either during gastrulation or immediately following gastrulation, these echinoids and ophiuroids develop larval skeleton from a population of mesenchyme cells separate from the adult/late mesenchymal cells. In holothuroids and ophiuroids, these cells derive from the tip of the archenteron or adjacent coeloms. In echinoids, the PMCs arise from small micromeres at the vegetal pole. From there, the cells migrate to the ventrolateral cluster and form the larval arms in echinoids and ophiuroids. The GRN governing skeletogenesis in these cells is distinct, although it shares a reliance on expression of Alx1 and Ets1, two key transcription factors in the skeletogenic GRN. Asteroid mesenchymal cells do not express Alx1 until expansion of the coeloms prior to rudiment development, likely accounting for the potential loss of skeletogenesis in larval asteroids (Koga et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Larval skeletogenesis in ophiuroids, echinoids, and holothuroids is a result of a similar GRN to the adult skeletogenic GRN being expressed early in development by mesenchyme cells arising during gastrulation (Gao \u0026amp; Davidson, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are two hypotheses of the origin of larval skeletogenesis; the first posits a single origin of adult skeletogenesis followed by multiple origins of larval skeletogenesis and convergent evolution of larval skeleton. This is owing to the phylogenetic placement of ophiuroids (which produce larval skeleton) as sister group to asteroids (which do not), as well as dissimilarities in the ophiuroid and echinoid larval GRN. The second hypothesis suggests that regardless of the sometimes contentious phylogenetic placement of ophiuroids (Cannon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pisani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Reich et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Telford et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), a single origin of larval skeletogenesis is more likely (Erkenbrack \u0026amp; Thompson, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), followed by a potential secondary loss of larval skeleton in Asteroidea. Echinoderm skeletogenic GRNs share specific characteristics, such as a conserved role for alx1, ets1, and vegfr, suggesting that differences in echinoid larval skeletogenesis and cell specification may be derived traits (Dylus et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Erkenbrack \u0026amp; Thompson, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; McCauley et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Both hypotheses indicate that adult skeletogenesis arose first, with a single origin. Given the shared origin of larval and adult skeletogenesis in echinoderms, and thyroid hormone regulation of larval and adult skeletogenesis in sea urchins (Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), we predict that TH regulation of skeletogenesis would be a shared feature of both larval and adult skeletogenesis in echinoids, ophiuroids, and asteroids.\u003c/p\u003e \u003cp\u003eIn this study, we seek to answer the question of whether thyroid hormones accelerate early and late skeletogenesis in two distantly related Echinoderms: the sea star \u003cem\u003eP. ochraceus\u003c/em\u003e, and the brittle star \u003cem\u003eO. aculeata\u003c/em\u003e, as well as to confirm previous results showing that thyroid hormones accelerate skeletogenesis in echinoids. We show that THs are capable of accelerating skeletogenesis in three classes of echinoderms, sea urchins, sea stars, and brittle stars (echinoids, asteroids, and ophiuroids). THs bind to cells proximal to sites of skeletogenesis and induce MAPK phosphorylation and skeletogenesis. These processes can be fully or partially inhibited by a competitive ligand of RGD-binding integrins, and by an inhibitor of MAPK activity. We discuss our findings in the context of the origin and evolution of TH regulation and the evolution of skeletogenesis in echinoderms.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal Care\u003c/h2\u003e \u003cp\u003e \u003cem\u003eOphiopholis aculeata\u003c/em\u003e were collected in Cobscook Bay, Maine, and shipped to Williamsburg, VA to be spawned (2020\u0026ndash;2021). Embryos were shipped overnight on ice to the University of Guelph (ON, Canada) as fertilized eggs in the case of experiments on gastrulae, or 6 days post-fertilization (dpf) in the case of the late-larval experiments. On arrival, ophiuroid larvae were transferred to 2L beakers at a density of 0.5-1 larva/mL. The cultures were cleaned manually and had the water replaced with filtered (0.2 \u0026micro;m) and UV-treated fresh artificial seawater (Instant Ocean) three times weekly. At the same time, cultures were fed \u003cem\u003eRhodomonas salina\u003c/em\u003e (UTEX LB 2763) at a density of 2000 cells/mL and \u003cem\u003eDunaliella tertiolecta\u003c/em\u003e (UTEX LB 999) at 3000 cells/mL. Gastrulae were collected for skeletogenesis assays at 2\u0026ndash;3 dpf. Late pluteus larvae were collected for skeletogenesis assays at 27 dpf.\u003c/p\u003e \u003cp\u003eWe collected Adult \u003cem\u003ePisaster ochraceus\u003c/em\u003e by hand during low tide at Friday Harbor, Washington (2019) and maintained them in seawater tables under flow-through seawater conditions (Hodin et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After spawning and fertilization, hatched embryos were transferred to 2L jars at a density of 1 larva per 2 mL. The cultures were cleaned manually and had the water replaced with fresh seawater three times weekly. At the same time, cultures were fed \u003cem\u003eRhodomonas sp.\u003c/em\u003e at a density of 3000 cells/mL and \u003cem\u003eDunaliella sp.\u003c/em\u003e at 4000 cells/mL. Gastrulae were collected after 48 hours at 11\u0026ndash;13\u0026deg;C and staged to ensure they were at early gastrulation (after ingression of primary mesenchyme cells and invagination of the blastopore, but before ingression of secondary mesenchyme cells and archenteron contact with the animal pole; see McClay et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e for review of gastrulation). Early bipinnaria were collected after 12 dpf, after coelomic pouch elongation. Late bipinnaria were collected at 22 dpf, just before formation of the brachiolaria arm buds.\u003c/p\u003e \u003cp\u003eAdult urchins (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e) were shipped from Monterey, CA (2019\u0026ndash;2023), where they were collected by diving and subsequently kept in tanks of artificial seawater at the Hagen Aqualab (University of Guelph, ON). The adults were fed a diet of kelp (\u003cem\u003eMacrocystis pyrifera\u003c/em\u003e, and \u003cem\u003eKombu spp\u003c/em\u003e.) every 2\u0026ndash;3 days. Temperature was maintained at 14\u0026deg;C and salinity at 31 ppt). Urchins were spawned by injecting 0.5\u0026ndash;1.5 mL of 0.5 M KCl in distilled water, depending on the size of the urchin. Sperm was collected dry by pipetting sperm directly from the gonopores. Females were inverted over a beaker of filtered artificial seawater (0.2\u0026micro;m \u0026ndash; FASW) to collect eggs. After spawning, eggs were washed twice with FASW. Diluted sperm (approx. 1:100) was titrated into the beaker of eggs until fertilization success reached at least 90%. Fertilized eggs were washed once more with FASW to remove excess sperm and allowed to develop at 12\u0026deg;C in a 2 L beaker until hatching. After 48 hours at 12\u0026ndash;14\u0026deg;C, hatched embryos were transferred to 2 L beakers at a density of 1 larva/mL. Sea urchin larval cultures were maintained at 14\u0026deg;C with salinity at 31\u0026ndash;33 g/L and larval density was gradually reduced on a weekly basis to an eventual density of approximately 250 larvae per liter. Cultures were stirred constantly using a paddle system previously described by Strathman and Strathman (1983) and kept on a 12:12 light cycle. The cultures were cleaned manually and had the water replaced three times weekly. At the same time, cultures were fed \u003cem\u003eRhodomonas salina\u003c/em\u003e at a density of 3000 cells/mL and \u003cem\u003eDunaliella tertiolecta\u003c/em\u003e at 4000 cells/mL.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDendraster excentricus\u003c/em\u003e (sand dollars) were collected from Crescent Beach, East Sound, Orcas Island in 2019. \u003cem\u003eD. excentricus\u003c/em\u003e were spawned by injecting 0.2-1.0 mL of 0.5 M KCl in distilled water, depending on the size of the animal. Sperm was collected dry by pipetting sperm directly from the gonopores. Females were inverted over a beaker of natural seawater to collect eggs. After spawning, eggs collected by pipette and distributed in a monolayer in a 500 mL beaker of seawater. Diluted sperm (approx. 1:100) was titrated into the beaker of eggs until fertilization success reached at least 90%. Fertilized eggs were washed once with seawater to remove excess sperm and allowed to develop at 11\u0026ndash;13\u0026deg;C in a 500 mL beaker until hatching. After 20 hours at 14\u0026deg;C, hatched embryos were transferred to 2L jars at a density of 1 larva/mL. Gastrulae were collected shortly thereafter and staged to ensure they were at early gastrulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Hormone preparation\u003c/h2\u003e \u003cp\u003eThyroid hormones (rT3, T4, T3) were prepared as described in Taylor and Heyland \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e (dissolved in filtered artificial seawater with 1% DMSO at a working concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M). PD98059 (inhibitor of MAPK ERK1/2; Sigma-Aldrich P215) and RGD peptide (inhibitor of integrin RGD binding pocket; Sigma-Aldrich A8052) were also used, and similarly diluted in filtered artificial seawater to a working concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M. Larvae were exposed to a final concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M rT3, T4, T3, or RGD peptide, or to 5 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M PD98059.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Skeletogenesis assays\u003c/h2\u003e \u003cp\u003eSkeletogenic assays were conducted as described in Taylor and Heyland (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, 2023). We scored the presence of skeletal spicules in repeated measures of individually isolated live larvae and determined the proportion of individuals which had developed skeleton (hourly for gastrulae, daily for late-stage larvae). Presence of skeletal spicules was confirmed with polarized light microscopy. Experiments were considered complete when at least one group asymptotically reached skeletogenesis (over 95% of at least one group achieved skeletogenesis), typically over the course of 4 hours for gastrulae, or 3\u0026ndash;5 days for late-stage larvae (S.p. gast.: 4 hr, S.p. larvae:, P.o. gast.: 72 hr, P.o. larvae: 120 hr, D.e. gast.: 4 hr, O.a.: gast: 5 hr, O.a. larvae: 96 hr). Larvae were kept at a density of 1 larva/mL in individual wells (24 well plate, 1mL/well). Larvae were placed on a slide for observation and skeletal structures were detected by polarized light microscopy on a compound microscope with inserted polarizing filter. Echinoderm skeletons are composed of a single birefringent crystal and polarized light microscopy is extremely suitable for detection of even initial skeletal spicules. In the case of gastrulae, skeleton was detected and scored at the ventrolateral clusters. In the case of late-stage larvae, the presence of adult skeleton developing in and around the rudiment was scored. Any skeleton which was not part of the larval arms was considered to be adult skeleton (Heyland \u0026amp; Hodin, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fluorescent Microscopy\u003c/h2\u003e \u003cp\u003eFluorescently labeled thyroxine (RHT4) was synthesized as described in Taylor and Heyland (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and used to determine T4 binding locations. RHT4 was prepared at a stock concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M and diluted in PBS to a final concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M for staining fixed samples. M8159 (antibody for phosphorylated ERK1/2; mouse monoclonal, Sigma-Aldrich M8159) was used to quantify MAPK (ERK1/2) phosphorylation (1:250). Hoechst 33342 (working stock 1 mg/mL, final concentration 5 \u0026micro;g/mL) was used to stain and identify nuclei. Thyroid hormones were diluted in filtered artificial seawater with DMSO (5:95 DMSO:FASW) to a working stock concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M before being diluted to a final concentration of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M in exposure conditions.\u003c/p\u003e \u003cp\u003eLarvae or gastrulae were exposed to 100 nM T4, 500 \u0026micro;M PD98059, 100 nM RGD peptide, 100 nM rT3, 100 nM RGD\u0026thinsp;+\u0026thinsp;100 nM T4 or a vehicle control (FASW with 0.0005% DMSO) for 90 minutes. Subsequently, they were fixed in 2% formaldehyde for 10 minutes at room temperature and washed with methanol and placed for 5 minutes in a -20\u0026deg;C freezer. Samples were washed 3x with phosphate-buffered saline with 0.1% Tween-20 (PBST; 5 minutes between washes) at room temperature, and blocked with 1% Goat Serum in PBST for 60 mins. Samples were exposed to primary MAPK antibody (M8159 1:250) for 12 hours overnight at 4\u0026deg; C. They were then washed 5x in PBST (5 minutes between washes) and exposed to secondary antibody for 4 hours (1:1000). This was followed by 7x washes in PBS (5 minutes between washes). Finally, samples were exposed to a mixture of Hoechst 33342 (5 \u0026micro;g/mL) and RHT4 (10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M) in PBS for 15 minutes, followed by 3x washes in PBS and were then mounted for imaging in DABCO/glycerol (see similar protocol in Wynen et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As the previously reported magnitude of TH effects on MAPK phosphorylation was over 3-fold increase in intensity (Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), a sample size of 4\u0026ndash;6 confocal images was predicted to be sufficient statistical power to test the effect of T4 on MAPK phosphorylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eMAPK phosphorylation was quantified by automatic cell counting in the Hoechst 33342 channel, followed by a measurement of fluorescent intensity of each individual cell in a radius around each nucleus (see S1 for details and S5 for ImageJ script). Intensity was normalized to the Hoechst 33342 signal to account for attenuation (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Fluorescent intensity of the top 5% of cells was compared between experimental groups and the control by one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. Intensities and means are reported as \u0026plusmn;\u0026thinsp;standard error. Colocalization of Hoechst nuclear stain, MAPK phosphorylation, and T4-binding locations in juvenile rudiments and midgut in late-stage larvae was measured with Pearson\u0026rsquo;s Coefficient using JACoP (v2.0; Bolte \u0026amp; Cordeli\u0026egrave;res, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the skeletogenic assays, experimental groups were compared with binary logistic regression of skeletal presence (binomial) and time (hours or days) contrasted to the control group. Additionally, spicule proportion over time was transformed to a rate of skeletogenesis (spicules/hour or spicules/day) and compared to the control with one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc. Proportions and means are reported as \u0026plusmn;\u0026thinsp;standard error.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Thyroid hormone effects on skeletogenesis\u003c/h2\u003e \u003cp\u003eThyroid hormones (T4, T3) accelerate skeletogenesis in a sea urchin (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e), a brittle star (\u003cem\u003eOphiopholis aculeata\u003c/em\u003e), and a sea star (\u003cem\u003ePisaster Ochraceus\u003c/em\u003e) (Fig.\u0026nbsp;3), as well as a sand dollar (\u003cem\u003eDendraster excentricus\u003c/em\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and as previously shown in Heyland and Hodin 2004). However, THs were not found to induce skeletogenesis in \u003cem\u003ePisaster ochraceus\u003c/em\u003e (sea star) gastrulae (n\u0026thinsp;=\u0026thinsp;20, ANOVA not possible as no groups produced skeleton; p\u0026thinsp;=\u0026thinsp;1.00). THs, including T4 and T3, accelerated onset of juvenile skeletogenesis (n\u0026thinsp;=\u0026thinsp;6, F(7,40)\u0026thinsp;=\u0026thinsp;36.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in sea star bipinnaria. The effect of THs on sea star bipinnaria was inhibited by PD9809 and RGD peptide (Tukey\u0026rsquo;s HSD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eTHs, including T4 and T3, accelerate skeletogenesis in \u003cem\u003eO. aculeata\u003c/em\u003e gastrulae (n\u0026thinsp;=\u0026thinsp;20, F(7,152)\u0026thinsp;=\u0026thinsp;12.45; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Tukey\u0026rsquo;s HSD, T4: 95%CI[1.75-fold, 2.69-fold], T3: 95%CI[1.08-fold, 2.03-fold]). RGD peptide (RGD) and PD98059 (PD) partially inhibit the effects of THs on skeletogenesis (Tukey\u0026rsquo;s HSD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In \u003cem\u003eO. aculeata\u003c/em\u003e larvae, THs (T4) accelerated juvenile skeletogenesis (n\u0026thinsp;=\u0026thinsp;12, F(3,44)\u0026thinsp;=\u0026thinsp;24.22; Tukey\u0026rsquo;s HSD p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This effect was partially inhibited by PD98059 (p\u0026thinsp;=\u0026thinsp;0.0163). The effect of RGD peptide on skeletogenesis was not tested in late-stage \u003cem\u003eO. aculeata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn all cases, T4 at 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M had a stronger effect than T3 at 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M (1.22-fold [S.p. 8-armed pluteus] to 3.71-fold [S.p. gastrula], x̄ = 2.31-fold). This difference was statistically significant in all cases except late-stage \u003cem\u003eS. purpuratus\u003c/em\u003e (8-armed pluteus stage; Tukey\u0026rsquo;s HSD, alpha\u0026thinsp;=\u0026thinsp;0.05). Gastrulae exposed to T4 showed rapid skeletogenesis (\u0026lt;\u0026thinsp;5h; Fig.\u0026nbsp;3M) in the ventrolateral clusters (Fig.\u0026nbsp;3A-B, E-F) in sea urchin and brittle stars, but not sea stars (Fig.\u0026nbsp;3I-J). This acceleration of skeletogenesis occurred over the course of 5 hours observation, shifting the onset of skeletogenesis forward by 1.05 h (\u0026plusmn;\u0026thinsp;0.07 h) in \u003cem\u003eS. purpuratus\u003c/em\u003e (sea urchin) gastrulae and 1.12 h (\u0026plusmn;\u0026thinsp;0.13 h) in \u003cem\u003eO. aculeata\u003c/em\u003e gastrulae. This represents a significant increase in the rate of skeletogenesis in T4-exposed gastrulae (\u003cem\u003eS. purpuratus\u003c/em\u003e: 3.13-fold, \u0026plusmn; 0.26; \u003cem\u003eO. aculeata\u003c/em\u003e: 2.22-fold, \u0026plusmn; 0.31).\u003c/p\u003e \u003cp\u003eTHs accelerated skeletogenesis in older larvae of all three classes of Echinoderms (sea urchin, sea star, and brittle star; Fig.\u0026nbsp;3N). Sea urchin larvae displayed increased skeletogenesis in the developing rudiment, as well as extra-rudiment skeleton such as juvenile spines and genital plates. Over the course of seven days of observation, T4-exposed larvae developed skeleton in the juvenile rudiment 2.78 days in advance of the control group (1.70-fold increased rate, \u0026plusmn; 0.14) and had an average rudiment skeletal development stage of 8.4 compared to the control group at 0.7 (staging scheme from Heyland \u0026amp; Hodin, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eT4 accelerated juvenile skeletogenesis of brittle star ophioplutei (23 d) in the terminal and radial plates adjacent to the gut and somatocoel (Fig.\u0026nbsp;3H). T4 brought forward the mean initiation of juvenile skeletogenesis by 0.96 d, resulting in a 1.79-fold (\u0026plusmn;\u0026thinsp;0.29) increase in the rate of skeletogenesis relative to the control. At the end of observation (27 d, 4 d post-exposure), every larva had begun juvenile skeletogenesis.\u003c/p\u003e \u003cp\u003eT4 drastically accelerated skeletogenesis of sea star late bipinnaria/early brachiolaria larvae (24 d). After 24 hours of T4 exposure, \u003cem\u003eP. ochraceus\u003c/em\u003e bipinnaria larvae produced skeletal spicules (oral plates) adjacent to the gut and somatocoel (Fig.\u0026nbsp;3L). By day 3, every single T4-exposed larva had produced skeleton, including oral and madreporitic plates, while not a single control larva had begun skeletogenesis (Fig.\u0026nbsp;3K). At the end of observation (29 d, 5 d post-exposure), 5/30 (\u0026plusmn;\u0026thinsp;2.7) of control larvae had produced skeleton. This represents a 4-day acceleration of skeletogenesis by T4, and a 22.6-fold (\u0026plusmn;\u0026thinsp;5.7) increase in the rate of skeletogenesis over the observed period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Thyroid hormone signaling mechanisms\u003c/h2\u003e \u003cp\u003eRGD peptide (a competitive inhibitor of the RGD-binding pocket on RGD-binding integrins) inhibited T4 acceleration of larval skeletogenesis in \u003cem\u003eD. excentricus\u003c/em\u003e (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), \u003cem\u003eS. purpuratus\u003c/em\u003e (Fig.\u0026nbsp;4A), and \u003cem\u003eO. aculeata\u003c/em\u003e gastrulae (Fig.\u0026nbsp;4C), as well as juvenile skeletogenesis in \u003cem\u003eP. ochraceus\u003c/em\u003e late bipinnaria (Fig.\u0026nbsp;4E; binomial logistic regression, n\u0026thinsp;=\u0026thinsp;12\u0026ndash;40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PD98059, an inhibitor of MAPK phosphorylation (ERK1/2), inhibited the effect of T4 on skeletogenesis in \u003cem\u003eS. purpuratus\u003c/em\u003e and \u003cem\u003eO. aculeata\u003c/em\u003e gastrulae (Fig.\u0026nbsp;4AC), as well as on juvenile skeletogenesis in \u003cem\u003eP. ochraceus\u003c/em\u003e, \u003cem\u003eS. purpuratus\u003c/em\u003e, and \u003cem\u003eO. aculeata\u003c/em\u003e late-stage larvae (Fig.\u0026nbsp;4D-F; binomial logistic regression, n\u0026thinsp;=\u0026thinsp;12\u0026ndash;40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eP. ochraceus\u003c/em\u003e gastrulae did not produce skeleton either with or without thyroid hormone exposure and as such there were no differences in rate of skeletogenesis between any \u003cem\u003eP. ochraceus\u003c/em\u003e gastrulae treatment groups (Fig.\u0026nbsp;4B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. T4 binding locations and colocalization with induced MAPK phosphorylation\u003c/h2\u003e \u003cp\u003eThyroid hormone binding increased MAPK phosphorylation in \u003cem\u003eS. purpuratus\u003c/em\u003e gastrulae (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RGD peptide alone had no statistically significant effect on MAPK phosphorylation, but did inhibit the effect of T4 (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). THs (T4) bound to PMCs in sea urchin gastrulae (Fig.\u0026nbsp;5B), as well as to regions near the tip of the developing archenteron. MAPK activation was seen most strongly in secondary mesenchyme cells (neural precursors) and the coelomic pouches (rudiment precursors), as well as to a lesser degree in PMCs ingressing at the vegetal pole. Both MAPK activation and T4 binding locations appeared to be localized to the membrane and cytoplasm (Fig.\u0026nbsp;5). Typically, the site of MAPK phosphorylation was colocalized with T4 binding sites within the cell (Fig.\u0026nbsp;5C\u0026prime;\u0026prime;-C‴, D\u0026prime;\u0026prime;-D‴) in the SMCs and PMCs, but not in the coelomic pouches. We did not detect any clear signs of nuclear binding in our confocal images.\u003c/p\u003e \u003cp\u003eT4 did not significantly increase MAPK phosphorylation in \u003cem\u003eP. ochraceus\u003c/em\u003e gastrulae (t[8]\u0026thinsp;=\u0026thinsp;0.526, p\u0026thinsp;=\u0026thinsp;0.61; Fig.\u0026nbsp;6E). RGD peptide decreased MAPK phosphorylation (t[8]\u0026thinsp;=\u0026thinsp;2.80, p\u0026thinsp;=\u0026thinsp;0.023; Fig.\u0026nbsp;6E). No distinct T4 binding sites were detected in either the control (Fig.\u0026nbsp;6A,C) or T4-exposed larvae (Fig.\u0026nbsp;6B,D). MAPK phosphorylation was most active in the developing coelomic pouches and was asymmetrical in all samples (20/20).\u003c/p\u003e \u003cp\u003eT4 bound primarily to the midgut, hindgut, and somatocoel in sea star bipinnaria (Fig.\u0026nbsp;7) and brachiolaria (Fig.\u0026nbsp;8). T4-exposed larvae displayed higher levels of MAPK phosphorylation in both bipinnaria and brachiolaria (t(10)\u0026thinsp;=\u0026thinsp;3.42, p\u0026thinsp;=\u0026thinsp;0.007; Fig.\u0026nbsp;8B; \u003cem\u003eP. ochraceus\u003c/em\u003e). In contrast, RGD peptide exposure resulted in a decrease in MAPK phosphorylation as well as inducing whole-body muscle contractions (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Larvae exposed to both RGD peptide and T4 displayed no statistically significant difference in MAPK phosphorylation from the control group (t(10)\u0026thinsp;=\u0026thinsp;1.31, p\u0026thinsp;=\u0026thinsp;0.218).\u003c/p\u003e \u003cp\u003eIn bipinnaria, the region of strongest MAPK phosphorylation was the somatocoel (Fig.\u0026nbsp;7). T4 bound most strongly to the membrane of gut cells, predominantly at the cell-cell junctions, but also to the nucleus (Fig.\u0026nbsp;7C\u0026Prime;). MAPK phosphorylation in the gut was strongly colocalized to cells which bind T4 (Fig.\u0026nbsp;7C\u0026Prime;\u0026rsquo;). Few presumptive T4 binding sites were observed in the hydrocoel, although the adjacent midgut did bind T4 (Fig.\u0026nbsp;7B).\u003c/p\u003e \u003cp\u003eWe observed extensive MAPK phosphorylation and increased T4 binding-sites in older sea star brachiolaria (Fig.\u0026nbsp;8). Putative T4-binding cells in the hindgut adjacent to the extending somatocoel were numerous and intensely fluorescent. Unlike in the bipinnaria, we observed T4 binding sites in the hydrocoel. As well, T4-binding sites were occasionally observed in the membrane/cytoplasm but frequently colocalized well with nuclear stains (Fig.\u0026nbsp;8D,E; r̄(4)\u0026thinsp;=\u0026thinsp;0.715).\u003c/p\u003e \u003cp\u003eWe found that sea star gut displayed highly asymmetrical putative T4 binding locations, with increased quantity and intensity of binding sites in the midgut and foregut closer to the somatocoel (Fig.\u0026nbsp;10). We observed possible T4-binding sites predominantly in the basal cell membrane of gut epithelia, as well as the membrane of gut-adjacent mesenchyme cells. Many of these cells also display binding in the nucleus, albeit to a lesser degree (r̄ = 0.566). There are more T4 binding cells adjacent to the somatocoel and the intensity of T4-binding appears to be higher adjacent to the somatocoel. In T4-exposed larvae, these locations colocalize approximately with increased MAPK phosphorylation in the gut, and to a lesser degree, the somatocoel and mesenchyme cells adhering to the gut/somatocoel boundary (t(10)\u0026thinsp;=\u0026thinsp;3.594, p\u0026thinsp;=\u0026thinsp;0.0049; Fig.\u0026nbsp;10B) relative to control larvae (Fig.\u0026nbsp;10A). RGD peptide prevents the effect of T4 on MAPK phosphorylation (t(10)\u0026thinsp;=\u0026thinsp;3.16, p\u0026thinsp;=\u0026thinsp;0.010), but potentially increases the number of T4-binding locations we observed (p\u0026thinsp;=\u0026thinsp;0.023; Fig.\u0026nbsp;10C).\u003c/p\u003e \u003cp\u003eIn late-stage ophiuroid larvae (eight-armed ophiopluteus), T4 bound to the gut, somatocoel, and presumptive skeletogenic mesenchyme in posterolateral arms (Fig.\u0026nbsp;9). In the posterolateral arms, we detected T4 binding sites predominantly in mesenchymal cells adhering to the skeletal rod (Fig.\u0026nbsp;9D). Most skeleton-adhering T4-binding cells displayed increased MAPK phosphorylation in response to T4-exposure. T4 significantly increased MAPK phosphorylation in \u003cem\u003eO. aculeata\u003c/em\u003e larval arms and somatocoel (t(6)\u0026thinsp;=\u0026thinsp;2.594, p\u0026thinsp;=\u0026thinsp;0.041; Fig.\u0026nbsp;9C). The effect of T4 on MAPK phosphorylation is potentially inhibited by RGD peptide with a mean decrease of 17% MAPK phosphorylation fluorescent intensity, although this difference was not statistically significant (t(6)\u0026thinsp;=\u0026thinsp;1.08, p\u0026thinsp;=\u0026thinsp;0.320; Fig.\u0026nbsp;9C).\u003c/p\u003e \u003cp\u003eWe imaged larvae with a variety of hydrocoel developmental stages, from the beginning of metamorphic development until the point at which the hydrocoel begins to wrap around the gut. A representative T4-exposed larva at the relatively advanced 5-lobed stage is depicted in Fig.\u0026nbsp;9 (B,E). No larvae examined displayed a high degree of T4-binding in the hydrocoel. In contrast, the somatocoel of most larvae presented with presumptive T4-binding sites, and T4-exposed samples showed a greater intensity of MAPK phosphorylation in the somatocoel, colocalized with T4-binding sites (Fig.\u0026nbsp;9F; r̄(6)\u0026thinsp;=\u0026thinsp;0.748).\u003c/p\u003e \u003cp\u003eIn sea urchin (\u003cem\u003eS. purpuratus\u003c/em\u003e) eight-armed pluteus larvae (rudiment soft tissue stages v-vii; Heyland \u0026amp; Hodin, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) RHT4 bound and fluoresced to gut cells, indicating potential T4-binding sites (as in the other echinoderms we examined). Additionally, T4 bound to the rudiment; primarily to the rapidly-developing layer most distal to the gut. In T4-exposed larvae, MAPK phosphorylation was induced in the gut and rudiment, strongly colocalized with putative T4-binding sites (r̄(6)\u0026thinsp;=\u0026thinsp;0.862). A close examination of binding sites in the T4-exposed larvae showed T4 binding in both the nucleus and cell membrane (Fig.\u0026nbsp;10B). Nuclear binding was primarily observed in the rudiment (r̄(6)\u0026thinsp;=\u0026thinsp;0.847), compared to the gut (r̄(6)\u0026thinsp;=\u0026thinsp;0.459). This is in contrast with \u003cem\u003eS. purpuratus\u003c/em\u003e gastrulae and control late-stage plutei which both showed binding primarily in the cell membrane and cytoplasm (Fig.\u0026nbsp;5B-D).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe found that THs (T4 and T3) accelerated skeletogenesis in larvae of distantly related echinoderm groups (sea stars, brittle stars, and sea urchins), suggesting a conserved regulatory mechanism of skeletogenic mesenchyme by THs. T4 bound to cells near sites of skeletogenic activity and increased MAPK phosphorylation. Both the acceleration of skeletogenesis and the MAPK phosphorylation was inhibited by RGD peptide (a competitive inhibitor of RGD-binding integrins), as well as PD98059 (an inhibitor of MAPK [ERK1/2] phosphorylation) in all species. These results provide further evidence for a role of nongenomic TH signaling via an integrin membrane receptor-mediated MAPK cascade and suggests a conserved regulatory mechanism between these groups.\u003c/p\u003e \u003cp\u003eOn a subcellular level, THs bind to the membrane and nucleus in echinoderms. We found nuclear binding to be more prominent in older echinoderm larvae, especially in \u003cem\u003eP. ochraceus\u003c/em\u003e brachiolaria midgut and \u003cem\u003eS. purpuratus\u003c/em\u003e rudiment nuclei. This coincides with evidence suggesting genomic transcriptional regulation is much more prominent in sea urchin eight-armed plutei relative to gastrulae (Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and reports that TH levels rise in older larvae as they develop to metamorphosis (Chino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e). THs accelerate sea urchin rudiment development (Chino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), suggesting that the rudiment may be a primary site of genomic TH signaling and regulation of metamorphosis.\u003c/p\u003e \u003cp\u003eWe found that sea star bipinnaria showed the greatest acceleration of skeletogenesis by thyroid hormones in the late larval stages, with THs inducing skeletogenesis weeks in advance of typical development (e.g. Pia et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This might be attributed to the early presence of coelomic mesenchyme adjacent to the gut, relative to \u003cem\u003eS. purpuratus\u003c/em\u003e and \u003cem\u003eO. aculeata\u003c/em\u003e, where late mesenchymal cells responsible for skeletogenesis don\u0026rsquo;t arise until rudiment formation (Gao \u0026amp; Davidson, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAsymmetric binding of thyroid hormones to gut cells is common to sea urchins, sea stars, and brittle stars. This may reflect the asymmetric development of adult structures in pre-metamorphic echinoderms. A greater quantity and intensity of the putative binding sites in the gut wall nearest the somatocoel suggests a potential shared mechanism of signaling from the gut wall to developing adult structures in the early rudiment. TH exposure resulted in acceleration of skeletogenesis in the rudiment, a process blocked by inhibitors of TH binding to RGD-binding integrins. This provides preliminary evidence that TH signaling via a membrane integrin receptor in gut and rudiment cells may regulate skeletogenesis in adjacent mesenchyme.\u003c/p\u003e \u003cp\u003eAnother possibility is that THs in the gut are binding to membrane transporters. Exogenous TH uptake has been proposed for echinoderms (Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Heyland \u0026amp; Moroz, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Miller \u0026amp; Heyland, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and gut wall transporters would be a crucial element in exogenous hormone uptake (Miller \u0026amp; Heyland, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The transporter hypothesis would partially explain why THs bind to some non-mesenchymal cells. These leave us with several explanatory hypotheses for this phenomenon: either THs are binding to transporters in the gut, or to receptors in the gut triggering release of a secondary signal (likely endocrine or neural, e.g. VEGF or serotonergic neuronal signaling).\u003c/p\u003e \u003cp\u003eThe arrangement of sea urchin hydrocoel and somatocoel is also unique within echinoderms. Sea urchin hydrocoel is layered on the somatocoel, while in most echinoderms the hydrocoel and somatocoel develop in distinct regions adjacent to the gut, with the hydrocoel developing proximal to the mouth region and the somatocoel developing closer to the midgut. In sea urchins, both somatocoel and hydrocoel develop adjacent to the midgut (M. M. Smith et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wessel et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This rearrangement of developing tissues in sea urchins may be related to the early skeletal development in the hydrocoel and the presence of TH binding sites and acceleration of development of sea urchin hydrocoel by THs.\u003c/p\u003e \u003cp\u003eSea urchin hydrocoel forms skeleton during rudiment development, but sea star and brittle star hydrocoel does not. Similarly, THs bind to sea urchin hydrocoel; particularly to the tips of the developing tube feet where skeleton will form. T4 shows little to no binding to sea star and brittle star hydrocoel. This suggests a possible link between TH receptor expression in sea urchin rudiment hydrocoel and TH regulation of skeletogenesis in sea urchin rudiment, which may be a synapomorphy of sea urchins relative to other echinoderms. Additionally, T4 activates skeletogenesis in \u003cem\u003eP. ochraceus\u003c/em\u003e before the coelomic sacs have wrapped around the gut, implying the source of skeletogenic cells is either migratory mesenchymal cells or gut cells, rather than the coelomic sac which will eventually form the hydrocoel.\u003c/p\u003e \u003cp\u003eCocurullo et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) provided circumstantial evidence based on transcriptional data that in sea urchin pluteus larvae, THs may be synthesized in the gut and neurons proximal to the gut (putative TH synthesis enzymes colocalized with typical gut and neuronal markers). They found expression of peroxidasin and deiodinase (both are potentially involved in TH synthesis), putative TH transporters, and both THR and integrin membrane receptor in early pluteus larvae. We found that the putative TH synthesis enzymes were regulated by THs in pre-metamorphic sea urchin larvae (Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This corresponds well with previous models in which THs were both exogenously and endogenously sourced; consumed as part of a typical algal diet and synthesized by iodinating dityrosine residues (Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Heyland \u0026amp; Moroz, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In both cases, THs would be sourced proximal to the gut. Given the lack of a circulatory system and hypothalamic-pituitary-thyroid axis, we predicted that TH binding sites in Echinoderm larvae might also be gut-proximal. As the juvenile rudiment is adjacent to the gut in species of Echinoderm which produce one, this would provide a plausible explanation for thyroid hormone regulation of development to metamorphosis, and settlement.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Evolution of skeletogenic gene regulation in echinoderms\u003c/h2\u003e \u003cp\u003eWhile the gene regulatory network (GRN) underlying adult skeletogenesis is an apparent apomorphy shared by extant echinoderms, larval skeletogenesis is likely a synapomorphy of sea stars, brittle stars, and sea urchins. (Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A possible model for the evolution of echinoderm larval skeletogenesis is of a single transfer of the adult GRN to early larval echinoderm development, followed by a secondary loss of larval skeleton in sea stars, and partial loss in sea cucumbers (Erkenbrack \u0026amp; Thompson, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Alternatively, multiple origins of skeletogenesis in Echinodermata have been proposed and this hypothesis remains under investigation (Cary \u0026amp; Hinman, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dylus et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McCauley et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMAPK phosphorylation of Ets1 is responsible for the majority of MAPK effects in skeletogenic sea urchin mesenchyme (Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and a MAPK cascade phosphorylating Ets1 is necessary for skeletogenesis in sea urchin larvae (Fernandez-Serra et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Khor \u0026amp; Ettensohn, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rafiq et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; R\u0026ouml;ttinger et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). During skeletogenesis, MAPK phosphorylates Ets1, leading to activation and increased transcription of Alx1, followed by expression and activation of skeletogenic products. For echinoderm skeletogenesis to occur, three elements are necessary: 1) A source of MAPK/ERK signal activation, 2) Ets1 expression and phosphorylation, 3) Alx1 expression. Ancestrally, Ets1 likely specified both adult skeleton and larval mesoderm in echinoderms (Koga et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results from this study confirm previous work on MAPK activation via THs in sea urchins (Taylor and Heyland \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and provide new evidence for this mechanism in sea stars, and brittle stars. These findings support the hypothesis that an integrin receptor binding THs and capable of MAPK signal transduction is a common feature of echinoderm skeletogenic mesenchyme. Recent work further supports this hypothesis, as T4 and to a lesser extent T3 bind to membrane fractions of sea urchin embryos (Taylor et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A putative RGD-binding integrin which is a likely candidate (Integrin αPβG) has been reported to have high expression in sea urchin gastrula PMCs, the skeletogenic cells during early sea urchin development (Cocurullo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Susan et al. 2000, Marsden and Burke 1997). Expression of RGD-binding proteins is crucial for the epithelial-mesenchyme transition in echinoderms (Hertzler \u0026amp; McClay, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Katow, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), vertebrates (Eliceiri \u0026amp; Cheresh, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ludwig et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nieberler et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and non-bilaterians (Magie \u0026amp; Martindale, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and may predate multicellular life (Custodio et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). RGD-binding of integrins to an extracellular matrix allows for selective adhesion, detection of the ECM, and signal transduction, which may have contributed to the evolution of multicellularity. Sponges and corals\u0026ndash;some of the earliest branching metazoans\u0026ndash;utilize iodinated tyrosine residues in the construction of the skeletal matrix, which in some sponges and corals is composed of up to 10\u0026ndash;26% iodine (dry weight), predominantly in the form of iodinated scleroproteins (Roche \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1952\u003c/span\u003e, Goldberg 1978). The greatest fraction of the iodinated tyrosine is in the form of T2 and T4 (Roche \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1952\u003c/span\u003e, Nowak et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) with exposure to T4 increasing the skeletal deposition rate in corals (Kingsley et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Nowak et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is therefore conceivable that thyroid hormones served as a structural element of early metazoan ECM. It is not clear whether T4 in corals has a regulatory effect, or if increased skeletal deposition rate as a result of T4 exposure is a consequence of increased material availability. In contrast, the primary active form of THs in vertebrates, T3, has not been detected in non-bilaterians (Tarrant, 2005, Roche \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1952\u003c/span\u003e). We speculate that the capability of RGD-binding integrins to bind T4 and other iodinated tyrosine compounds may have an ancient origin in non-bilaterians (discussed in Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), though the mechanism of action and function of T4 in non-bilaterians remains unknown.\u003c/p\u003e \u003cp\u003eEchinoderms possess a single THR gene, orthologous to THRβ, and while binding of THs to the echinoderm THR has not been demonstrated, we have previously shown that T4 can regulate gene expression of genes proximal to TH response elements in the genome (Taylor et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, T4 can bind to nuclear extract from echinoid cells (Saito et al. 2012). Our results here show TH binding to the nucleus in sea star and sea urchin gut and rudiment. We only observed this binding in late-stage larvae, corroborating our previous hypothesis that T4 regulation of gene expression via THR is a late-larval phenomenon in sea urchins (Taylor et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, T4 exposure resulted in an increase of TH binding locations, an effect which was not inhibited by RGD peptide in sea urchin, ophiuroids and sea star larvae. Autoregulation of the THR is a classic sign of canonical TH signaling via a nuclear receptor, an effect which we also detected in Taylor et al. (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since RGD is not known to bind to the nuclear hormone receptor, this suggests that expression of TH binding locations may be under control of a non-integrin mechanism, such as via autoregulation by the THR, providing evidence for canonical TH signaling activity in all three classes of Echinoderm we examined.\u003c/p\u003e \u003cp\u003eThe commonality of binding locations and the evidence that THs may regulate metamorphic development in echinoids (Chino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Saito et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and sea stars (Johnson and Cartright 1996) suggests a potential role for THR in regulating echinoderm metamorphosis. However, unpublished data mentioned in Holzer et al. (2017) suggests that T4, among other TH compounds, may not bind to sea star isolated THR, and these authors hypothesized that THR may be functioning while unliganded. Additionally, it appears that retinoic acid signaling via the retinoic acid receptor (RAR) instead may be a mechanism for nuclear hormone receptor-regulated metamorphosis of sea stars, as in crinoids (Yamakawa et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Evidence for genomic regulation of development by THs by binding to a THR is strong in sea urchins and tenuous for sea stars and brittle stars. Future research will have to test this hypothesis further by analyzing transcriptomic response to THs (as in Taylor et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), ideally with a ChIP assay or binding kinetics to confirm TH binding to THR.\u003c/p\u003e \u003cp\u003eThyroid hormone signaling has been repeatedly co-opted to regulate developmental processes. Not only do THs work via multiple independently evolved mechanisms (Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), THs regulate developmental processes which also evolved independently. For example, THs appear to regulate skeletogenesis in both echinoderms and chordates \u0026ndash; but echinoderm skeletogenesis is a novelty of the phylum and distinct in both regulatory mechanisms and material components from chordate skeletogenesis (Ben-Tabou de-Leon, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Livingston et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Murdock, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rafiq et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). THs additionally regulate metamorphic development or settlement in molluscs, annelids, echinoderms, and chordates (Carpizo-Ituarte \u0026amp; Rosa-Velez, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chino et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994b\u003c/span\u003e; Fukazawa et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Holzer, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Johnson, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Klootwijk et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Paris et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Saito et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), a process which may or may not share a single origin. As well, THs regulate diverse other developmental systems, including neurogenesis, vasculogenesis, metabolism, and myogenesis (Brent, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). THR actions rely on RXR, a transcription factor which is already implicated in a large number of transcriptional regulatory events (Evans \u0026amp; Mangelsdorf, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and creates a signaling complex with THR. The genomic TH mechanism has the ability to activate and regulate a long list of genes (Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and RXR is ubiquitously expressed (Evans \u0026amp; Mangelsdorf, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Viera-Vera \u0026amp; Garc\u0026iacute;a-Arrar\u0026aacute;s, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), meaning coexpression of THR and TH availability are the main regulators of cell responsiveness to THs via the genomic pathway. Synthesis and metabolism of THs is common to most metazoans, as most classes, with the notable exception of insects, have the ability to synthesize thyroxine and other THs (Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nuclear hormone receptor heterodimers with RXR as a component are implicated in control of development and metamorphosis in metazoans (Hall \u0026amp; Thummel, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Laudet, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), including cnidarians which do not possess a THR (Fuchs et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These features combined create a readily evolvable system of TH gene regulation. Similarly, non-genomic signaling acts via integrin activation of MAPK, a ubiquitous pathway which can be triggered by a commonly expressed class of proteins: RGD-binding integrins (Bergh et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Cody et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Davis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Receptiveness to integrin mediated signaling can easily evolve in multiple cell types, relying only on proximity to a source of THs and expression of a TH-binding integrin making TH signaling a versatile system in both development and evolution. In a cell containing both signaling pathways, we speculate that external THs would first activate the integrin signaling pathway, triggering rapid phosphorylation and activation of proteins (including the nuclear THR) before being transported to the cytoplasm/nucleus and binding to the nuclear THR to regulate gene transcription.\u003c/p\u003e \u003cp\u003eThyroid hormone regulation of skeletogenic mesenchyme appears to be a common mechanism in echinoderms, including conserved expression of an integrin receptive to T4 which can trigger a MAPK cascade, and a conserved requirement for a MAPK cascade phosphorylating Ets1 and initiating skeletogenesis via Alx1 (Czarkwiani et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gao \u0026amp; Davidson, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Koga et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McCauley et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; R\u0026ouml;ttinger et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Skeletogenesis is an essential component of metamorphic development and acceleration of skeletogenesis may play a role in regulation of metamorphosis, particularly in echinoid larvae (Heyland \u0026amp; Hodin, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Parks et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Taylor \u0026amp; Heyland, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wray \u0026amp; Raff, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). TH levels in sea urchins rise prior to metamorphosis (Chino et al., 1994), contributing to the control of developmental timing of metamorphosis and settlement. The acceleration of skeletogenesis by THs represents an extremely evolvable mechanism, as THs would already be present in and proximal to the rudiment, and skeletogenic mesenchyme would already be sensitive to MAPK phosphorylation. MAPK regulation of skeletogenic activity seems to be present in all echinoderms. The only necessary element would be expression of a TH-binding integrin to trigger or enhance MAPK signaling. TH regulation of skeletogenesis appears to exist in sea urchins, sea stars, and brittle stars. Alternatively, the inverse could apply: TH regulation of metamorphosis would be extremely evolvable in the case that THs are already synthesized or transported to the rudiment to control skeletogenesis. THs accelerate both larval and adult skeletogenesis in larval brittle stars and sea urchins (and adult skeletogenesis in sea star larvae). It seems increasingly likely that TH regulation of echinoderm skeletogenesis is at least as ancient as the divergence of Echinozoa and Asterozoa. Critically, the hypothesis that endogenous hormone synthesis is responsible for regulation of skeletogenesis in larval and adult echinoderms is still mostly untested. TH mechanisms in echinoid metamorphic development may involve peroxidase-facilitated diffusion or transport (Heyland et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Miller \u0026amp; Heyland, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), but the ultimate source of thyroid hormones in embryonic skeletogenesis remains unclear. Additionally, responsiveness to THs in echinoderm gastrulae may be a consequence of a shared GRN in gastrulae, larvae, and adults, and not a trait which is active under normal physiological conditions. Future work should focus on inhibition of TH synthesis and attempt to determine which effects of THs, if any, are derived from endogenous hormone synthesis versus exogenous hormone sources (as in the vitamone hypothesis; Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), as well as the source of thyroid hormones in Echinoderm embryos and larvae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Skeletal loss/reduction in sea stars and sea cucumbers\u003c/h2\u003e \u003cp\u003eIn the case of a single larval origin of skeletogenesis, there is currently no satisfactory explanation for the loss of skeleton in sea star larvae. Sea cucumbers and sea stars have likely undergone an independent loss of larval skeleton in comparison to sea urchins and brittle stars. The reduction in sea cucumber larval skeleton might be attributed to the same evolutionary process as the reduction in adult skeleton, given that they share a GRN (McCauley et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and expression of biomineralization-related genes is greatly reduced in the adult (A. B. Smith \u0026amp; Reich, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is possible that the adult skeleton of sea stars has also been reduced, as they possess less skeleton (dry weight) than either sea urchins or brittle stars (Dubois, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The adult skeleton is highly reduced in sea cucumbers, some crinoids, and some extinct echinoderms (Zamora et al. 2022, Smith and Reich \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Smirnov 2017). In contrast with sea star larvae, sea cucumbers still initiate larval skeletogenesis and have larval skeletogenic mesenchyme. It is likely that the sea cucumber larval skeleton was reduced from an ancestral state, given the close relation to sea urchins and brittle stars, both of which have extensive larval skeletons. It has been suggested that sea cucumber adult skeletal reduction is an example of paedomorphosis (Cu\u0026eacute;not, 1948; Smirnov, 2015); however, it is not clear whether reduction in larval or adult skeletogenesis came first, or if due to the shared regulatory mechanism, the reduction was simultaneous. The secondary reduction in sea cucumber skeleton appears to be a likely result of modification or loss of transcription factors in the skeletogenic mesenchyme, possibly related to the reduction in adult skeleton. This contrasts with reduced sea star skeleton which relies on non-expression of the skeletogenic transcription factors until metamorphic development. For this reason, we hypothesize that sea cucumber larvae may still be responsive to thyroid hormone exposure and predict that they might undergo accelerated or expanded skeletogenesis after TH exposure. This could be tested in future experiments and would shed light on both sea star and sea cucumber skeletogenesis, as well as the differing evolutionary trajectories that led to reduction/loss of larval skeletogenesis in both lineages.\u003c/p\u003e \u003cp\u003eSea star larvae do not express Alx1 and are therefore incapable of producing skeleton until metamorphic development when coelom-derived mesenchyme likely becomes primed for skeletogenesis by expressing Alx1 and other skeletogenic regulatory genes (Koga et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In contrast, sea cucumbers retain expression of Alx1, but have lost expression of downstream secondary transcription factors in the skeletal mesenchyme (McCauley et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This explains why sea star gastrulae were unresponsive to THs in our experiments: Alx1 is not present to be phosphorylated and therefore no skeleton can be formed (Koga et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The full complement of skeletogenic regulator genes is present and functional in sea stars, but not expressed in larval cells until the production of skeleton in the rudiment prior to metamorphosis. This may represent a secondary loss of the previously evolved heterochronic activation of the skeletogenesis program during larval gastrulation (Shashikant et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Thyroid hormone regulation of developmental timing\u003c/h2\u003e \u003cp\u003eRegulation of developmental timing is critically important in the case of both larval and juvenile skeletogenesis. Larval skeleton in sea urchins and brittle stars supports the larval arms which act as both feeding structures and a defence against predation (Boidron-Metairon, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Strathmann et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Skeletogenesis must begin soon after hatching, as the larvae prepare to feed. In late-stage larvae, development to metamorphosis and juvenile skeletogenesis are essential steps to prepare for life on the benthos. \u003cem\u003eP. ochraceus\u003c/em\u003e can produce skeleton weeks before metamorphosis (Pia et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eS. purpuratus\u003c/em\u003e, develops skeletal elements 5\u0026ndash;7 days prior to metamorphosis, and \u003cem\u003eO. aculeata\u003c/em\u003e produces skeleton only one or two days before metamorphosis. These represent differing strategies of resource investment in juvenile structures during the larval feeding stage. Control of resource allocation towards metamorphic development must be carefully balanced with larval growth (Strathmann et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). For instance, in sea urchins, THs increase skeletogenesis in the juvenile rudiment while shortening the larval arms (Armstrong \u0026amp; Lessios, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e; Heyland et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wynen et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, THs may allow for control of resource investment in both brittle star and sea star larvae. One potential source of THs is exogenous (Eales, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Heyland \u0026amp; Moroz, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), implying that food availability might modulate late larval development.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. ochraceus\u003c/em\u003e is also notable for spending a longer than typical time developing juvenile structures as a brachiolaria larva (Pia et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The early responsiveness of \u003cem\u003eP. ochraceus\u003c/em\u003e skeletogenesis to THs may relate to the need for temporal control of the extended development to metamorphosis. In contrast, \u003cem\u003eO. aculeata\u003c/em\u003e develops few skeletal structures prior to a rapid metamorphosis and was only responsive to TH acceleration of juvenile skeletogenesis several days before a typical metamorphosis would occur.\u003c/p\u003e \u003cp\u003eWe did not examine any direct-developing echinoderms (echinoderms which develop directly from egg to adult without a feeding larval stage). Echinoderm eggs are maternally provisioned with THs (Chino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e) and TH regulation of metamorphic development is sufficient to allow an obligate feeding species with an indirect development to act as a facultative feeding larva and metamorphose without the need for food (Heyland et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), albeit with a much smaller juvenile size. Based on the potentially shared mechanisms and role of THs in sea stars and brittle stars, we predict that the same phenomenon might be observed in non-echinoid echinoderms.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThyroid hormones, principally thyroxine, accelerate skeletogenesis in sea urchins, sea stars, and brittle star larvae. THs also accelerate skeletogenesis in brittle star and sea urchin gastrulae, but do not induce ectopic skeletogenesis in sea star gastrulae (which normally do not produce skeleton). Thyroid hormones bind to cells proximal to regions of skeletogenesis, primarily in the gut and rudiment, and stimulate MAPK phosphorylation. RGD peptide, an inhibitor of the RGD binding pocket in RGD-binding integrins, inhibits the effect of thyroid hormones in all three echinoderm classes examined. PD98059, an inhibitor of MAPK signaling, prevents the effect of THs on skeletogenesis, especially in sea star larvae. Thyroid hormones may act non-genomically, via a membrane integrin receptor-mediated MAPK cascade in sea stars, brittle stars, and sea urchins. TH regulation of mesenchyme cell activity may be an ancient mechanism to control timing of development, including skeletogenesis. TH regulation of skeletogenesis in late mesenchyme cells prior to metamorphosis may have been co-opted to regulate larval skeletogenesis in sea urchins and brittle stars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conception, design, interpretation, and revisions of this manuscript. E.T. wrote the manuscript draft, conducted the experiments, and analyzed the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArmstrong, A. F., \u0026amp; Lessios, H. A. (2015). 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The sea cucumber genome provides insights into morphological evolution and visceral regeneration. \u003cem\u003ePLOS Biology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(10), e2003790. https://doi.org/10.1371/journal.pbio.2003790\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"developmental-biology-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evod","sideBox":"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)","snPcode":"13227","submissionUrl":"https://submission.nature.com/new-submission/13227/3","title":"Developmental Biology Advances","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3858209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3858209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThyroid 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 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. 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 skeletogenesis in the three echinoderm groups. Additionally, we detect thyroid hormone binding to the cell membrane and nucleus during metamorphic development in echinoderms. 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. Additionally, 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.\u003c/p\u003e","manuscriptTitle":"Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 06:04:56","doi":"10.21203/rs.3.rs-3858209/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-23T18:56:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-12T20:37:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78fd3c6e-5e84-48f8-a0d6-416fe14caaf1","date":"2024-02-03T05:01:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"694c57eb-7793-48b2-8f8b-129cd7e13a9b","date":"2024-01-29T21:17:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-27T23:40:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-16T13:55:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-16T13:55:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"EvoDevo","date":"2024-01-12T21:14:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"developmental-biology-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evod","sideBox":"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)","snPcode":"13227","submissionUrl":"https://submission.nature.com/new-submission/13227/3","title":"Developmental Biology Advances","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccf03977-9e81-43a4-b3f6-fe2dfee46baa","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:09:38+00:00","versionOfRecord":{"articleIdentity":"rs-3858209","link":"https://doi.org/10.1186/s13227-024-00226-2","journal":{"identity":"developmental-biology-advances","isVorOnly":false,"title":"Developmental Biology Advances"},"publishedOn":"2024-08-07 15:57:47","publishedOnDateReadable":"August 7th, 2024"},"versionCreatedAt":"2024-01-18 06:04:56","video":"","vorDoi":"10.1186/s13227-024-00226-2","vorDoiUrl":"https://doi.org/10.1186/s13227-024-00226-2","workflowStages":[]},"version":"v1","identity":"rs-3858209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3858209","identity":"rs-3858209","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}