Table 1.
Oligonucleotide primers used for RTqPCR and chromatin immunoprecipitation (ChIP) assay.
Fig 1.
RNA-sequencing analysis in tadpole brain during metamorphosis.
A. Pie charts with pairwise comparisons of differentially expressed genes (DEGs) between four stages of metamorphosis. Numbers in red areas of the pie charts represent genes upregulated in Stage B, while the numbers in the blue areas of the pie charts represent genes downregulated in Stage B. Gene regulation changes increased as metamorphosis progressed, with roughly half the genes upregulated and half downregulated. Xenopus illustrations © Natalya Zahn (2022), source Xenbase (www.xenbase.org RRID:SCR_003280) [34]. B. Expression patterns of four known TH-regulated genes in tadpole brain (preoptic area/thalamus/hypothalamus) analyzed at four Nieuwkoop-Faber (NF) stages of spontaneous metamorphosis. Plotted are data from an RNA-seq experiment (n = 3/NF developmental stage). thrb–thyroid hormone receptor b; dio3 –monodiodinase type 3; thibz–thyroid hormone induced bZip protein; klf9 –Krüppel-like factor 9. All except dio3 have been shown to be directly regulated by TH-TR [35–39], which is also supported by the current TR ChIP-seq experiment. C. Heatmaps showing expression changes for the top 100 upregulated and the top 100 downregulated genes (determined during the developmental interval NF50-NF62; S1 Table).
Table 2.
Top twenty genes with annotation upregulated from premetamorphosis (NF50) to metamorphic climax (NF62) in tadpole brain.
Table 3.
Top twenty genes with annotation downregulated from premetamorphosis (NF50) to metamorphic climax (NF62) in tadpole brain.
Fig 2.
Patterns of gene regulation in X. tropicalis tadpole brain during spontaneous metamorphosis or after T3 treatment analyzed by RNA-sequencing.
A. Clustering analysis showing five patterns of gene expression changes across four stages of metamorphosis. B. Venn diagram showing numbers of genes regulated with overlaps in three developmental stage comparisons. C. Venn diagram showing the overlap between all genes that changed expression during spontaneous metamorphosis with genes induced or repressed by exogenous T3 in premetamorphic tadpoles.
Table 4.
Comparisons of gene counts from RNA-sequencing experiments.
Fig 3.
Distribution across the genome of thyroid hormone receptor ChIP-seq peaks in X. tropicalis neural cells.
A. TR ChIP-seq peaks were found across the entire genome and uniformly distributed on the 10 chromosomes of X. tropicalis. B. A majority of TR ChIP-seq peaks were located +/- 1 kb from the transcription start sites (TSS) of genes. C. Pie chart showing the distribution of TR ChIP-seq peaks across the X. tropicalis genome by genomic feature. D. IGV genome browser tracks showing the locations of TR ChIP-seq peaks (TR peaks; light blue bars) at four loci. Shown are two genes that were upregulated (nucleus accumbens associated 1—nacc1, peak range 0–291; thyroid hormone induced bZip protein—thibz, peak range 0–250) and 2 that were downregulated (aurora kinase A—aurka, peak range 0–326; E2F transcription factor 5—e2f5, peak range 0–233) during metamorphosis. Gene structures are shown in dark blue below the genome tracks.
Table 5.
Accounting of TR ChIP-seq peaks.
Fig 4.
Cell cycle control genes and genes encoding components of the Wnt/b-catenin signaling pathway are downregulated during metamorphosis.
A. Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data (top) and RTqPCR data (bottom; n = 5/NF developmental stage) for four cell cycle control genes: Cyclin b1—ccnb1.2; cyclin a2—ccna2; cyclin-dependent kinase 1—cdk1; E2F transcription factor—e2f1. B. KEGG pathway analysis of RNA-seq data for gene expression changes during metamorphosis in X. tropicalis tadpole brain. Shown is the cell cycle control pathway. C. Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data for 4 genes: Wnt family member 1—wnt1; Wnt family member 3—wnt3; frizzled class receptor 2—fzd2; frizzled class receptor 10—fzd10.
Fig 5.
Genes encoding stem/progenitor cell markers are downregulated, while genes encoding neural differentiation-related proteins are upregulated during metamorphosis.
A. Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data for 6 stem/progenitor cell marker genes: Vimentin—vim; nestin—nes; notch 1 receptor—notch1; hairy and enhancer of split 5 gene 4 –hes5.4; hairy and enhancer of split 5 gene 3—hes5.3; hairy and enhancer of split 6 gene 2—hes6.2. B. Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data for 12 neural differentiation-related genes: Neuronal differentiation 6—neurod6; bone morphogenetic protein 1—bmp1; cholinergic receptor muscarinic 2—chrm2; transient receptor potential cation channel, subfamily M, member 8—trpm8; sodium channel, voltage gated, type V alpha subunit—scn5a; monoamine oxidase A—maoa; glutamate receptor, ionotropic, N-methyl-D-aspartate 3B - grin3b; calcium/calmodulin dependent protein kinase ID—camk1d; calmodulin 1—calm1; purinergic receptor P2Y, G-protein coupled, 1—p2ry1; purinergic receptor P2X, ligand gated ion channel, 5—p2rx5; myelin basic protein—mbp.
Fig 6.
Genes encoding proteins involved with ribosome biogenesis and protein synthesis are downregulated during metamorphosis.
A. KEGG pathway analysis of RNA-seq data for gene expression changes during metamorphosis in X. tropicalis tadpole brain (preoptic area/thalamus/hypothalamus). Shown is the ribosome biogenesis pathway. B. Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data for 9 genes: Eukaryotic translation elongation factor 1 alpha 1—eef1a1o; eukaryotic translation elongation factor 1 beta 2—eef1b2; eukaryotic translation elongation factor 1 alpha 1—eef1a1; ribosomal protein L9—rpl9; ribosomal protein L3—rpl3; ribosomal protein S19- rps19; ribosomal oxygenase 2- riox2; eukaryotic translation initiation factor 3 subunit E—eif3e; eukaryotic translation initiation factor 3 subunit D—eif3d.
Fig 7.
Genes encoding neuroendocrine-related proteins are upregulated during metamorphosis.
Shown are the mean+SEM (n = 3/NF developmental stage) of RNA-seq count data for 12 genes: Nuclear receptor subfamily 3 group C member 2 (mineralocorticoid receptor)—nr3c2; arginine vasotocin—avt*; inhibin subunit beta B—inhbb; prolactin, gene 1—prl.1; somatostatin receptor 2—sstr2; insulin like growth factor binding protein 2—igfbp2; melanocortin 4 receptor—mc4r; galanin receptor 1—galr1; gonadotropin releasing hormone 1—gnrh1; gonadotropin releasing hormone receptor 2—gnrhr2; steroidogenic acute regulatory protein—star; estrogen receptor 1—esr1. *This gene is mislabeled in the genome database as arginine vasopressin (avp) which is a mammalian gene. The amphibian gene is arginine vasotocin (avt).