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BACKGROUND: The thyroid hormones-thyroxine (T4) and 3,5,3'triiodothyronine (T3)-regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS-sorted NSPCs in response to T3 during Xenopus laevis metamorphosis.
RESULTS: We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3-upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome.
CONCLUSION: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis.
Figure 1. Isolation and characterization of EGFP− and EGFP+ cells from the zGFAP::EGFP transgenic tadpoles treated with T3. (A) Stage 52 tadpoles were exposed to control (untreated) and 5 nMT3 solutions for 16 hours. The whole brain was isolated and disaggregated with Papain and individual EGFP− and EGFP+ cells were sorted by cytometry. Total RNA was isolated from unsorted cells, EGFP− cells and EGFP+ cells. (B) RT-qPCR for egfp in EGFP− cells (E−) and EGFP+ cells (E+) of RNA samples isolated from untreated and T3-treated tadpoles. The expression of egfp was normalized to eef1a1 and compared to the E− condition in four independent biological replicates and plotted as log2-expression. ***P < 0.001, ANOVA with Sidak's multiple comparisons test between E− and E+ for control and T3 conditions. (C–E) The expression of klf9 (C), thrβA (D) and nrep (E) was analyzed by RT-qPCR in isolated brain EGFP+ cells from control and T3-treated tadpoles. The expression of each gene was normalized to eef1a1 and compared to the control condition in 3 (nrep) or 4 (klf9 and thrβA) independent biological replicates and plotted as log2-expression. *P < 0.05, **P < 0.01, unpaired Student's t-test.
Figure 2. Differential expression analysis of control and T3 transcriptomes in NPSCs. Volcano plot (âlog10 adjusted P-value vs log2 fold-change) for the comparison between untreated and T3 libraries. Blue dots represent differential expression genes found using DESeq2 (adjusted P-valueâ<â0.05). Selected gene names are included (See text for details)
Figure 3. T3 increases the expression of chac1, nfic and mat2a after 16 hours in the tadpolebrain. RTqPCR was performed in whole brain samples of control tadpoles and T3-treated tadpoles for klf9 (A) as a positive control of T3 induction, and chac1 (B), nfic (C), and mat2a (D), genes induced by T3 in NSPCs after 16 hours. The expression of each gene was normalized to eef1a1 and compared to the control condition in 2 (nrep) or 3 (klf9 and thrβA) independent biological replicates and plotted as log2-expression. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student's t-test
Figure 4. Gene Ontology (GO) analysis of T3-upregulated genes. Bubble plots of enriched categories of T3-upregulated genes for Biological Processes (BP) (A), molecular function (MF) (B), cellular component (CC) (C) and KEGG pathway analysis (D). Color represents the âlog10 (P-value) and the size of the bubble represent the number of genes for each category. GO and KEGG pathways analysis were performed with DAVID resources and plotted using SRplot
