Mengeling BJ et al. (2022), Retinoid-X receptor agonists increase thyroid h...

XB-ART-58900

PLoS One 2022 Apr 13;174:e0266946. doi: 10.1371/journal.pone.0266946.

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Retinoid-X receptor agonists increase thyroid hormone competence in lower jaw remodeling of pre-metamorphic Xenopus laevis tadpoles.

Mengeling BJ , Vetter LF , Furlow JD .

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Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, we've used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development.

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Species referenced: Xenopus laevis
Genes referenced: aurkb mmp11 mmp13l rpl8 runx2 thibz thrb
GO keywords: thyroid hormone mediated signaling pathway [+]

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	Fig 1. RXR agonists potentiate T3-induced changes to lower jaw morphology, while an RXR antagonist abrogates T3 action. a-h: Representative dorsal head photos of tadpoles treated for five days starting at 1wk-PF (as indicated: DMSO, 10 nM T3, and 30 nM Bex, 1 nM TBT, 600 nM UVI with or without T3). i-k: Quantification of changes to the LJ angle. Boxes represent 25th-75th percentiles with the line at the median (n = 10–15 from 2–3 clutches), and whiskers represent the min and max values. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (****, p < 0.0001). l: Effect of Bex and TBT on T3-induced LJ angle changes as a function of time. Data points represent means from 20 animals from two different clutches; error bars delineate the 95% confidence intervals, indicating statistical significance. m: Treatment with 30 nM Bex augments LJ angle narrowing as a function of T3 dose. Statistics are the same as in the time course, although the clutches were different.
	Fig 2. RXR agonists potentiate T3-induced remodeling of LJ cartilage and resorption of gill cartilage. Representative ventral head photos of tadpoles treated for five days starting at 1wk-PF with vehicle (DMSO, a), 10 nM T3 (b), 10 nM T3 + 30 nM Bex (c), or 10 nM T3 + 1 nM TBT (d) and then stained with Alcian blue to visualize cartilage. MC, Meckel’s cartilage; IR, infracostal cartilage; CH, ceratohyal cartilage; and BA, branchial arches. e-f: Meckel’s cartilage lengths do not change with treatment. g-h: Changes in LJ angle measured from the end of the MC to the middle of the IR to the end of the opposite MC. Box plots are as in Fig 1 (n = 10 from 2 clutches of five per treatment. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; *, p < 0.001).
	Fig 3. RXR agonists potentiate T3 action on cellular proliferation in the LJ of 1wk-PF tadpoles. a: MC-IR region used for quantitation of proliferation. b-f: Representative photos of the effects of different treatments on proliferation using phopho-Ser10-H3 reactivity. g-i: Quantification of proliferation in the presence and absence of T3 and RXR ligands normalized to the area counted. Boxes and statistics are as in Fig 1 (n = 20–30 jaws from 2–3 clutches). j-l: RXR ligands do not significantly affect the T3-induced expression of aurora kinase B mRNA (aurkb). Bars represent the mean of 3–6 independent clutches, and statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.01; , p < 0.05).
	Fig 4. RXR ligands have gene-specific effects on T3-induced differential gene expression. Left column: The effect of RXR agonist Bex on T3-induced gene expression. Middle column: The effect of environmental RXR agonist TBT on T3-induced genes. Right column: The effect of RXR antagonist UVI on T3-induced genes. Striped bars indicate the presence of the RXR ligand, and white bars show induction in the absence of the RXR ligand. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.01; , p < 0.05).
	Fig 5. RXR agonists potentiate T3 action in the LJ in pro-metamorphic NF 54 tadpoles. a. Bex potentiates the T3-induced decrease in the LJ angle in NF 54 tadpoles treated for three days. Boxes and statistics are as in Fig 1 (n = 14 jaws from 3 clutches). Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.05). b. TBT potentiates T3-inducible, integrated luciferase reporter expression in the LJ of NF 54 tadpoles.

References [+] :

Bassett, Role of Thyroid Hormones in Skeletal Development and Bone Maintenance. 2016, Pubmed

	Fig 1. RXR agonists potentiate T3-induced changes to lower jaw morphology, while an RXR antagonist abrogates T3 action. a-h: Representative dorsal head photos of tadpoles treated for five days starting at 1wk-PF (as indicated: DMSO, 10 nM T3, and 30 nM Bex, 1 nM TBT, 600 nM UVI with or without T3). i-k: Quantification of changes to the LJ angle. Boxes represent 25th-75th percentiles with the line at the median (n = 10–15 from 2–3 clutches), and whiskers represent the min and max values. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (****, p < 0.0001). l: Effect of Bex and TBT on T3-induced LJ angle changes as a function of time. Data points represent means from 20 animals from two different clutches; error bars delineate the 95% confidence intervals, indicating statistical significance. m: Treatment with 30 nM Bex augments LJ angle narrowing as a function of T3 dose. Statistics are the same as in the time course, although the clutches were different.
	Fig 2. RXR agonists potentiate T3-induced remodeling of LJ cartilage and resorption of gill cartilage. Representative ventral head photos of tadpoles treated for five days starting at 1wk-PF with vehicle (DMSO, a), 10 nM T3 (b), 10 nM T3 + 30 nM Bex (c), or 10 nM T3 + 1 nM TBT (d) and then stained with Alcian blue to visualize cartilage. MC, Meckel’s cartilage; IR, infracostal cartilage; CH, ceratohyal cartilage; and BA, branchial arches. e-f: Meckel’s cartilage lengths do not change with treatment. g-h: Changes in LJ angle measured from the end of the MC to the middle of the IR to the end of the opposite MC. Box plots are as in Fig 1 (n = 10 from 2 clutches of five per treatment. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; *, p < 0.001).
	Fig 3. RXR agonists potentiate T3 action on cellular proliferation in the LJ of 1wk-PF tadpoles. a: MC-IR region used for quantitation of proliferation. b-f: Representative photos of the effects of different treatments on proliferation using phopho-Ser10-H3 reactivity. g-i: Quantification of proliferation in the presence and absence of T3 and RXR ligands normalized to the area counted. Boxes and statistics are as in Fig 1 (n = 20–30 jaws from 2–3 clutches). j-l: RXR ligands do not significantly affect the T3-induced expression of aurora kinase B mRNA (aurkb). Bars represent the mean of 3–6 independent clutches, and statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.01; , p < 0.05).
	Fig 4. RXR ligands have gene-specific effects on T3-induced differential gene expression. Left column: The effect of RXR agonist Bex on T3-induced gene expression. Middle column: The effect of environmental RXR agonist TBT on T3-induced genes. Right column: The effect of RXR antagonist UVI on T3-induced genes. Striped bars indicate the presence of the RXR ligand, and white bars show induction in the absence of the RXR ligand. Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.01; , p < 0.05).
	Fig 5. RXR agonists potentiate T3 action in the LJ in pro-metamorphic NF 54 tadpoles. a. Bex potentiates the T3-induced decrease in the LJ angle in NF 54 tadpoles treated for three days. Boxes and statistics are as in Fig 1 (n = 14 jaws from 3 clutches). Statistics show results from Sidak’s multiple comparison test in conjunction with 2-way ANOVA (**, p < 0.0001; , p < 0.001; , p < 0.05). b. TBT potentiates T3-inducible, integrated luciferase reporter expression in the LJ of NF 54 tadpoles.