Thyroid Hormone Receptor Alpha Is Required for Thyroid Hormone-Dependent Neural Cell Proliferation During Tadpole Metamorphosis

Abstract

Thyroid hormone (T₃) plays several key roles in development of the nervous system in vertebrates, controlling diverse processes such as neurogenesis, cell migration, apoptosis, differentiation, and maturation. In anuran amphibians, the hormone exerts its actions on the tadpole brain during metamorphosis, a developmental period dependent on T₃. Thyroid hormone regulates gene transcription by binding to two nuclear receptors, TRα and TRβ. Our previous findings using pharmacological and other approaches supported that TRα plays a pivotal role in mediating T₃ actions on neural cell proliferation in Xenopus tadpole brain. Here we used Xenopus tropicalis (X. tropicalis) tadpoles with an inactivating mutation in the gene that encodes TRα to investigate roles for TRα in mitosis and gene regulation in tadpole brain. Gross morphological analysis showed that mutant tadpoles had proportionally smaller brains, corrected for body size, compared with wildtype, both during prometamorphosis and at the completion of metamorphosis. This was reflected in a large reduction in phosphorylated histone 3 (pH3; a mitosis marker) immunoreactive (ir) nuclei in prometamorphic tadpole brain, when T₃-dependent cell proliferation is maximal. Treatment of wild type premetamorphic tadpoles with T₃ for 48 h induced gross morphological changes in the brain, and strongly increased pH3-ir, but had no effect in mutant tadpoles. Thyroid hormone induction of the direct TR target genes thrb, klf9, and thibz was dysregulated in mutant tadpoles. Analysis of gene expression by RNA sequencing in the brain of premetamorphic tadpoles treated with or without T₃ for 16 h showed that the TRα accounts for 95% of the gene regulation responses to T₃.

Keywords: Xenopus; development; knockout (KO); metamorphosis; neurogenesis; thyroid hormone (T3).

Figure 1

Brain size and cell proliferation is reduced in thra^mt−exon4 animals. We measured body length and brain volume in wild type (WT) and thra^mt−exon4 animals at two stages of development. Brain volume was measured on fixed brains as described in the Materials and Methods. Bars are the mean ± SEM. (A) Comparison of body length and brain volume of WT and thra^mt−exon4 prometamorphic tadpoles (NF stage 56; n = 6/genotype). (B) Comparison of body length and brain volume of WT and thra^mt−exon4 newly metamorphosed frogs (NF stage 66; n = 8/genotype). (C) Comparison of phosphorylated histone 3 (pH3) immunoreactive nuclei in the brain of WT and thra^mt−exon4 NF stage 56 tadpoles (n = 4–6/genotype). ^*p < 0.01, ^**p < 0.001, ^***p < 0.0001, unpaired Student's t-test.

Figure 2

Thyroid hormone receptor α is required for T₃-dependent morphological changes, and induction of cell proliferation in tadpole brain. We treated wild type (WT) and thra^mt−exon4 premetamorphic (NF stage 54) tadpoles with T₃ (5 nM) or vehicle added to the aquarium water for 42 h, then measured brain size and phosphorylated histone 3 immunoreactivity (pH3-ir) in the brain as described in the Materials and Methods. Bars are the mean ± SEM. (A) Exogenous T₃ caused dramatic morphological changes in the brains of WT, but not thra^mt−exon4 tadpoles. Shown are dorsal images of fixed tadpole brains. (B) Exogenous T₃ decreased length, and increased width of WT tadpole brain, but had no effect in thra^mt−exon4 animals (^***p < 0.0001, unpaired Student's t-test). In vehicle-treated tadpoles, the baseline brain length and width were both smaller in thra^mt−exon4 compared with WT (^###p < 0.0001, unpaired Student's t-test). (C) Treatment with T₃ strongly increased pH3-ir in WT (p < 0.0001, unpaired Student's t-test), but decreased it in thra^mt−exon4 tadpole brain (p < 0.0001). Shown is the fold change in total brain pH3-ir nuclei in each genotype. The dashed line indicates fold change of 1.0 = no change.

Figure 3

Gene transcription responses to exogenous T₃ are impaired in thra^mt−exon4 tadpole brain. We treated wild type (WT) and thra^mt−exon4 premetamorphic (NF stage 54) tadpoles with T₃ (5 nM) or vehicle added to the aquarium water for different times, then measured target gene mRNA levels in brain (region of the preoptic area/thalamus/hypothalamus) by RTqPCR using absolute quantification (for thra and thrb, panel A) or relative quantification (genes in panel B). (A) Thyroid hormone receptor α is the major TR subtype expressed in tadpole brain. Comparison of baseline mRNA levels, and effects of T₃ treatment for 42 h on thra and thrb mRNA levels in WT and thra^mt−exon4 tadpole brain. Bars represent the mean ± SEM (n = 4/genotype/treatment). Note that the baseline (vehicle-treated) thra mRNA level is 14 times greater than thrb mRNA in WT tadpole brain. The baseline thra mRNA was significantly lower, while the thrb mRNA was significantly higher in thra^mt−exon4 compared with WT tadpole brain (^##p < 0.001, Student's unpaired t-test). Treatment with T₃ had no effect on thra mRNA, but strongly induced thrb mRNA in both genotypes (^**p < 0.001, ^***p < 0.0001, Student's unpaired t-test). (B) Venn diagram representing the numbers of T₃-regulated genes determined by RNA-seq conducted on RNA isolated from brains of wild type and thra^mt−exon4 NF stage 54 tadpoles treated with or without T₃ for 16 h. (C) Time course of induction by T₃ in tadpole brain of mRNAs for three direct T₃ response genes, thrb, klf9, and thibz (note that these three genes are included in the 178 genes that overlap between WT and thra^mt−exon4). Points represent the mean ± SEM (n = 4/genotype/time point). The mRNA level for each of the genes was elevated at 8 h in both genotypes (p < 0.001, ANOVA), and the slope of each curve was significantly different between WT and thra^mt−exon4 (p < 0.0001, linear regression analysis).

Figure 4

RNA sequencing shows that the majority of gene regulation responses to exogenous T₃ are lost in thra^mt−exon4 tadpole brain. We treated wild type (WT) and thra^mt−exon4 premetamorphic (NF stage 54) tadpoles with T₃ (5 nM) added to the aquarium water for 16 h, then analyzed mRNA levels in brain (region of the preoptic area/thalamus/hypothalamus) by RNA-sequencing or RTqPCR using relative quantification. (A) Venn diagram representing the numbers of T₃-induced genes determined by RNA-sequencing conducted on RNA isolated from tadpole brains treated with or without T₃. (B) Validation of gene regulation for four T₃-induced genes found within the overlap between the WT and thra^mt−exon4 gene sets (109 genes). (C) Venn diagram representing the numbers of T₃-repressed genes determined by RNA-sequencing conducted on RNA isolated from tadpole brains treated with or without T₃. (D) Validation of gene regulation for four T₃-repressed genes found within the overlap between the WT and thra^mt−exon4 gene sets (69 genes). Bars represent the mean ± SEM (n = 4/genotype/treatment). For comparisons of vehicle with T₃ treated: ^*p < 0.01, ^**p < 0.001, ^***p < 0.0001, Student's unpaired t-test. For comparisons of baseline mRNA levels (vehicle treated) among the two genotypes: ^#p < 0.01, Student's unpaired t-test.

Figure 5

Effects of loss of TRα on baseline gene transcription in premetamorphic tadpole brain. We treated wild type (WT) and thra^mt−exon4 premetamorphic (NF stage 54) tadpoles with T₃ (5 nM) added to the aquarium water for 16 h, then analyzed mRNA levels in brain (region of the preoptic area/thalamus/hypothalamus; including the pituitary) by RNA-sequencing or RTqPCR using relative quantification. (A) Venn diagram representing the number of T₃-induced genes in WT and the number of genes whose baseline mRNA level was elevated in thra^mt−exon4 tadpoles, determined by RNA-sequencing conducted on RNA isolated from tadpole brains treated with or without T₃. (B) Validation of gene regulation for two T₃-induced genes found within the overlap between the T₃ induced WT and elevated baseline in thra^mt−exon4 gene sets (74 genes). (C) Venn diagram representing the number of T₃-repressed genes in WT and the number of genes whose baseline mRNA level was reduced in thra^mt−exon4 tadpoles, determined by RNA-sequencing conducted on RNA isolated from tadpole brains treated with or without T₃. (D) Validation of gene regulation for two T₃-repressed genes found within the overlap between the T₃ repressed WT and reduced baseline in thra^mt−exon4 gene sets (78 genes). Bars represent the mean ± SEM (n = 4/genotype/treatment). For comparisons of vehicle with T₃ treated: ^*p < 0.01, ^**p < 0.001, ^***p < 0.0001, Student's unpaired t-test. For comparisons of baseline mRNA levels (vehicle treated) among the two genotypes: ^#p < 0.01, Student's unpaired t-test.