Raj S et al. (2023), Metamorphic gene regulation programs in Xenopus...

XB-ART-60154

PLoS One 2023 Jan 01;186:e0287858. doi: 10.1371/journal.pone.0287858.

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Metamorphic gene regulation programs in Xenopus tropicalis tadpole brain.

Raj S, Sifuentes CJ, Kyono Y, Denver RJ.

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Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs that underlie morphogenesis. Gene expression screens using tissues from premetamorphic tadpoles treated with TH identified some TH target genes, but few studies have analyzed genome-wide changes in gene regulation during spontaneous metamorphosis. We analyzed RNA sequencing data at four developmental stages from the beginning to the end of spontaneous metamorphosis, conducted on the neuroendocrine centers of Xenopus tropicalis tadpole brain. We also conducted chromatin immunoprecipitation sequencing (ChIP-seq) for TRs, and we compared gene expression changes during metamorphosis with those induced by exogenous TH. The mRNA levels of 26% of protein coding genes changed during metamorphosis; about half were upregulated and half downregulated. Twenty four percent of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks. Genes involved with neural cell differentiation, cell physiology, synaptogenesis and cell-cell signaling were upregulated, while genes involved with cell cycle, protein synthesis, and neural stem/progenitor cell homeostasis were downregulated. There is a shift from building neural structures early in the metamorphic process, to the differentiation and maturation of neural cells and neural signaling pathways characteristic of the adult frog brain. Only half of the genes modulated by treatment of premetamorphic tadpoles with TH for 16 h changed expression during metamorphosis; these represented 33% of the genes whose mRNA levels changed during metamorphosis. Taken together, our results provide a foundation for understanding the molecular basis for metamorphosis of tadpole brain, and they highlight potential caveats for interpreting gene regulation changes in premetamorphic tadpoles induced by exogenous TH.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: aurka aurkb avp bmp1 calm1 camk1d cdc16 cdc27 cdk14 cdk15 cdk17 cdk5r2 cdkl5 cdkn1a chrm2 dio3 dock3 e2f5 eef1a1 eef1a1o eef1b2 eif3d eif3e esr1 fabp2 gadd45g gal galr1 gnrh1 gnrhr2 grin3b hba1 hbg1 hes4 hes5.3 hes5.4 hes6.2 hsd17b6 igfbp2 inhbb ins klf9 maoa mbp mc4r myc nacc1 nes neurod6 notch1 nr3c2 p2rx5 p2ry1 prl rgcc riox2 rpl3 rpl9 rps19 scn5a snap25 snap91 sstr2 star tet2 tet3 thibz thrb trpm8 vim
GO keywords: Wnt signaling pathway [+]

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	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).
	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.
	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.
	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).
	S1 Fig. Region of the tadpole brain dissected for RNA extraction and analysis by RNA-seq at four stages of metamorphosis, or after T3 treatment of premetamorphic tadpoles. The coronal anatomical diagrams of Xenopus brain are from Tuinhof and colleagues [93] with modifications by Yao and colleagues [94]. Dotted lines demarcate the region of the tadpole brain that was dissected. Abbreviations: Apl, Amygdala pars lateralis; BNST, bed nucleus of the stria terminalis; C, central thalamic nucleus; CeA, central amygdala; dp, dorsal pallium; LA, lateral amygdala; LH, Lateral hypothalamus lp, lateral pallium; Lpv, lateral thalamic nucleus, pars posteroventralis; MeA, medial amygdala; mp, medial pallium; NPv, nucleus of the paraventricular organ; nII, cranial nerve II; OB, olfactory bulb; OT, optic tectum; P, posterior thalamic nucleus; POa, preoptic area; Tel, telencephalon; VH, ventral hypothalamic nucleus; VM, ventromedial thalamic nucleus.

References [+] :

Abu-Jamous, Clust: automatic extraction of optimal co-expressed gene clusters from gene expression data. 2018, Pubmed