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Figure 1. Sea urchin indirect life history. Major stages of the indirect life cycle of S. purpuratus during which skeletogenesis occurs. Post-hatching, the embryo grows larval arms, becoming a free-swimming pluteus larva. The pluteus feeds on plankton in the water column, accumulating resources for metamorphosis. Prior to attaining metamorphic competence, the larva grows a juvenile rudiment with skeletal structures necessary for survival as a juvenile, including adult spines and tube feet. Also pictured is the metamorphosed juvenile, and the adult sea urchin.
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Figure 2. Regulation of skeletogenesis in sea urchins. Two main inputs are known to be necessary for skeletogenesis in sea urchins, VEGF secreted by ectodermal cells, and a MAPK (ERK1/2) cascade with an unknown trigger. ETS1 and ALX1 are transcription factors, and are important regulators of skeletogenesis in sea urchins, controlling almost half of the genes differentially expressed in primary mesenchyme cells (11). Their activity is necessary for skeletogenesis to occur. Both Ets1 and Alx1 are activated or upregulated by MAPK (ERK1/2).
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Figure 3. Thyroid hormones accelerate rate of skeletogenesis in gastrulae and pluteus larvae. (A) Gastrulae were exposed for 20 h, from 24 to 44 h post fertilization (n = 40–81). (B) Six armed larvae were exposed for 4 days, from 10 days post fertilization to 14 days post fertilization (n = 63). Larvae were scored at regular intervals for the presence or absence of skeletal spicules; either hourly (gastrulae) or daily (six armed larvae). Spicule deposition rate was normalized to the control. In both stages examined, T4, T3, and T2 accelerated skeletogenesis (p= <0.001–0.002) in a dose-dependent fashion (p = 0.001–0.045), while Triac inhibited skeletogenesis (p = 0.008). (Binary logistic regression with Bonferroni corrected p-values. *p < 0.05, **p < 0.01, ***p < 0.001, indicates significant difference from control group).
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Figure 4. Thyroxine accelerates rudiment development in S. purpuratus larvae. Pictured are late stage larvae kept either without T4 (A) or with 100 nM T4 for 5 days (B). T4 exposed larvae have significantly more developed skeletal elements in the rudiment, as well as shortened larval arms. Larvae at soft tissue stage 1 (C) or skeletal stage 0 (D) were exposed to T4 for 5 days [n = 12, for staging scheme, see (21)]. T4 drastically accelerated skeletogenesis in the rudiment (Mann–Whitney, p < 0.001) as well as accelerating other markers of metamorphic competence, including arm retraction and tube feet protrusion, but did not accelerate early soft tissue development (Mann–Whitney, p = 0.63). This suggests that the rudiment of late stage larvae may become responsive to T4 only as skeletal development begins. r, rudiment; t.f, tube foot.
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Figure 5. Four days exposure to thyroxine (100 nM) causes ectopic skeleton. Frequently, after exposure to high levels of THs, larvae develop skeletal abnormalities. (A) Skeletal rings, as well as a duplicate posterodorsal arm can be seen. (B) An ectopic spicule, as well as abnormal branching from the posterodorsal arm. (C) Several ectopic spicules. (D) A number of unusual skeletal protrusions on the post-oral arm, as well as presumptive primary mesenchyme cells in the process of laying down skeleton. (E) Only T4 and T3 caused skeletal abnormalities, with protrusions and duplicate posterodorsal arms being observed with higher exposure to T4 (Z-test, p < 0.001), where nearly every larvae examined had a skeletal aberration. Ectopic skeleton was significantly more present in all levels of T4 exposure and the higher level of T3 exposure (Z-test, p = < 0.001–0.004). No skeletal abnormalities were observed in the control or rT3 groups. d, duplicate posterodorsal arm; e, ectopic skeleton; p, skeletal protrusion; m, primary mesenchyme cell. Images were taken with differential interference contrast microscopy (DIC). Scale bars (A–C) (100 μm); (D) (20 μm). (*p < 0.05).
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Figure 6. Acute and chronic exposure to T4 have similar effects. In the acute exposure groups, 12 day old larvae were exposed to rT3 or T4 for 1 h before being washed thoroughly and placed in clean seawater (n = 21). The chronic exposure groups were exposed for 3 days (n = 82). Larvae from both groups were imaged daily, and the presence or absence of spicules in the developing posterodorsal arms was noted. (C) Acute exposure (1 h) to thyroxine has similar effects to chronic exposure over 4 days, with high levels of T4 causing a significant increase in spicule initiation in both groups (Z-test, p < 0.01). (A) representative larva from T4 treatment, (B) representative larvae from control and rT3 treatments.
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Figure 7. The effect of T4 on skeletogenesis is inhibited by PD98059, an ERK1/2 inhibitor but not p38 inhibitor. (A) Gastrulae were pre-exposed to PD98059 before being exposed to T4 for 20 h (n = 100). Following exposure, spicule initiation was monitored hourly for 5 h. The hypothesis that PD98059 inhibits the effect of T4 on skeletogenesis was tested using a binary logistic regression (D = 232, df = 110). T4 increased the rate of skeletogenesis, while PD98059 inhibited the effect of T4 on skeletogenesis (Bonferroni corrected p <0.0001). The highest levels of PD prevented skeletogenesis completely, an effect which was rescued by T4 (Bonferroni corrected p = 0.0012). This suggests that T4 acts through a MAPK (ERK1/2) cascade. (B) Gastrulae were pre-exposed to SB203580 before being exposed to T4 for 20 h (n = 20). Following exposure, spicule proportion at 41 hpf was observed. The hypothesis that SB inhibits skeletogenesis was tested using a binary logistic regression (D = 11, df = 12). T4 increased the rate of skeletogenesis (Bonferonni corrected p < 0.001), while SB had neither a significant effect on skeletogenesis nor any interaction with the effect of T4 (p = 0.996).
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Figure 8. Ets1 upregulation after T4 exposure is prevented by the presence of a MAPK (ERK1/2) inhibitor but skeletogenesis related genes are mostly downregulated in late stage larvae. (A) 90 min T4 exposure causes upregulation of Ets1, a regulator of skeletogenesis. This effect is blocked by PD98059, an inhibitor of MAPK. Each group is a single pooled sample of an estimated 1,000–2,000 gastrulae (24 hpf). Mean ΔΔCt with comparison to ubiquitin as a reference gene is displayed with standard error of technical replicates and unexposed larvae from the same culture as a control. (B) Larvae were exposed to 100 nM T4 for 24 h before being sampled. These data are taken from 8 pooled samples of 100 larvae each and are relative to an unexposed control group. Mean ΔΔCt with comparison to ubiquitin as a reference gene is displayed with standard error of technical replicates and unexposed larvae from the same culture as a control. Sm50, a marker of skeletogenic activity, is upregulated, while genes normally responsible for regulating skeletogenesis, Alx, and Ets, are downregulated. The genomic thyroid hormone receptor and VEGF receptor are also downregulated. Expression levels were calculated with reference to non-exposed controls.
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Figure 9. Colocalization of fluorescently labeled T4 (RH-T4—A), labeled rT3 (RH-rT3—D) with primary mesenchyme cells (PMCs). (B,E) Merged image showing colocalization of fluorescently labeled thyroid hormones and 6a9 antibody for PMCs. Gastrulae were incubated with RH-T4 and RH-rT3 for 30 min prior to fixation in methanol. Following fixation, immunohistochemistry with 6a9 antibody (C,F) was used to stain the membrane of PMCs. RH-T4 binds specifically to the membrane of PMCs, while RH-rT3 does not, suggesting a specific binding site for T4 in the PMCs.
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Figure 11. T4 exposure causes phosphorylation of MAPK. Gastrulae were exposed to T4 for 90 min prior to fixation and staining with an antibody that detects the phosphorylated form of ERK1/2 in sea urchins. Panels (A–C) show immunohistochemical results when embryos were exposed to T4 carrier control. Panels (D–F) show representative images that were taken at the same time and exposure when embryos were treated with 100 nM T4 (A,D: DIC image, B,E: 133 ms exposure MAPK detection, C,F: 400 ms exposure MAPK detection). (G) Quantification of the signal revealed that T4 (100 nM) exposed samples fluoresced with significantly higher intensity than the control (3.25 × intensity after subtracting background, 400 ms exposure; ANOVA n = 4, ***p < 0.001). All images are same scale–scale bar 20 μm. A, animal pole; V, vegetal pole.
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Figure 12. Genomic and non-genomic mechanisms of thyroid hormone action in vertebrates. (A) In the canonical genomic signaling, T4 is released by the thyroid gland, before being deiodinated into T3. T3 then binds to the nuclear thyroid hormone receptor (TR) in complex with the retinoid X receptor (RXR) at hormone response elements in the genome, regulating transcription. (B) In non-genomic signaling, T4/T3 can bind to an integrin membrane receptor. When binding to integrin αVβ3, T4/T3 triggers a MAPK cascade, resulting in the phosphorylation and activation of transcription factors. (C) Extranuclear TR is the other described receptor for non-genomic activity. T3 binds to cytoplasmic or membrane-bound TR, phosphorylating PI3K, as well as triggering Protein Kinase G (PKGII) or nitric oxide (NO) signaling pathways through the activation of cGMP and NO Synthase (NOS) respectively. See (30) for review of currently known non-genomic pathways.
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Figure 13. Proposed mechanism of thyroid hormone action in sea urchins (A) and potentially conserved system in vertebrates. (B) In sea urchins, thyroid hormones likely bind to an integrin on the surface of primary mesenchyme cells, triggering a MAPK cascade. MAPK phosphorylates Ets1 and Alx1, activating them. Ets1 and Alx 1 upregulate regulatory controls of skeletogenesis, leading to initiation of skeletogenesis as well as ingression of the primary mesenchyme cells; an epithelial to mesenchyme transition. A Tgif-Hex-Etg regulatory loop maintains the skeletogenenic fate of the cell and also promotes skeletogenesis. In vertebrates, thyroid hormones bind to integrin αVβ3 on the membrane of fibroblasts, triggering a MAPK cascade. MAPK phosphorylates Ets, promoting angiogenesis, and epithelial to mesenchyme transition via Alx. Some details differ, notably the lack of direct regulation of angiogenesis by Alx in the vertebrate mechanism, the absence of the Tgi-Hex-Erg regulatory loop, and the inhibition of VegfR by Hex.
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