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Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine-Dependent Neurogenesis.

Samarut E, Bekri A, Drapeau P - Front Mol Neurosci (2016)

Bottom Line: As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis.Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down.While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Research Center of the University of Montreal Hospital Center Montréal, QC, Canada.

ABSTRACT
How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signaling that promotes calcium transients in neural stem cells (NSCs) and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signaling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine.

No MeSH data available.


Differential expression analysis. (A) Venn diagram showing the total number of differentially expressed genes in each tests. Test#1 identified a total of 6971 differentially expressed genes (6569 + 187 + 215, p-value < 0.05) by comparing the transcriptome of NSCs from glra4a morpholino-injected (GlyR KD) versus uninjected embryos (WT). Test#2 identified 635 differentially expressed genes between the transcriptome of NSCs from control morpholino-injected (Ctrl KD) versus uninjected (WT) (233 + 215 + 187, p-value < 0.05). 402 genes (215 + 187) were overlapping between tests #1 and #2 (in blue). Test#3 compared the transcriptome of GlyR KD versus Ctrl KD for these 402 genes and identified 187 genes for which the differential expression was significant (p-value < 0.05). These genes were compiled with 6569 genes differentially expressed in GlyR KD (in red) and compose the final list of 6756 specific genes differentially expressed in NSCs upon GlyR KD (stripped red). (B) Volcano plot showing each of the 6756 genes from the final list established above. Blue dots represent the total 6756 specific genes filtered on a p-value < 0.05. Orange dots show the 1162 genes with a /log2FC/ > 1 and red dots show the 141 genes with a /log2FC/ > 2.
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Figure 3: Differential expression analysis. (A) Venn diagram showing the total number of differentially expressed genes in each tests. Test#1 identified a total of 6971 differentially expressed genes (6569 + 187 + 215, p-value < 0.05) by comparing the transcriptome of NSCs from glra4a morpholino-injected (GlyR KD) versus uninjected embryos (WT). Test#2 identified 635 differentially expressed genes between the transcriptome of NSCs from control morpholino-injected (Ctrl KD) versus uninjected (WT) (233 + 215 + 187, p-value < 0.05). 402 genes (215 + 187) were overlapping between tests #1 and #2 (in blue). Test#3 compared the transcriptome of GlyR KD versus Ctrl KD for these 402 genes and identified 187 genes for which the differential expression was significant (p-value < 0.05). These genes were compiled with 6569 genes differentially expressed in GlyR KD (in red) and compose the final list of 6756 specific genes differentially expressed in NSCs upon GlyR KD (stripped red). (B) Volcano plot showing each of the 6756 genes from the final list established above. Blue dots represent the total 6756 specific genes filtered on a p-value < 0.05. Orange dots show the 1162 genes with a /log2FC/ > 1 and red dots show the 141 genes with a /log2FC/ > 2.

Mentions: Between 65 and 89 million high quality reads per condition were sequenced and we were able to map about 86% of these reads onto the zv9 version of the zebrafish genome (ensemble release 77). Using DESeq2 algorithm, we assessed the differentially expressed genes comparing the dataset from GlyR knockdown NSCs with uninjected WT NSCs (test#1, Figure 3A) and found 6,971 differentially expressed genes (p-value < 0.05).


Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine-Dependent Neurogenesis.

Samarut E, Bekri A, Drapeau P - Front Mol Neurosci (2016)

Differential expression analysis. (A) Venn diagram showing the total number of differentially expressed genes in each tests. Test#1 identified a total of 6971 differentially expressed genes (6569 + 187 + 215, p-value < 0.05) by comparing the transcriptome of NSCs from glra4a morpholino-injected (GlyR KD) versus uninjected embryos (WT). Test#2 identified 635 differentially expressed genes between the transcriptome of NSCs from control morpholino-injected (Ctrl KD) versus uninjected (WT) (233 + 215 + 187, p-value < 0.05). 402 genes (215 + 187) were overlapping between tests #1 and #2 (in blue). Test#3 compared the transcriptome of GlyR KD versus Ctrl KD for these 402 genes and identified 187 genes for which the differential expression was significant (p-value < 0.05). These genes were compiled with 6569 genes differentially expressed in GlyR KD (in red) and compose the final list of 6756 specific genes differentially expressed in NSCs upon GlyR KD (stripped red). (B) Volcano plot showing each of the 6756 genes from the final list established above. Blue dots represent the total 6756 specific genes filtered on a p-value < 0.05. Orange dots show the 1162 genes with a /log2FC/ > 1 and red dots show the 141 genes with a /log2FC/ > 2.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Differential expression analysis. (A) Venn diagram showing the total number of differentially expressed genes in each tests. Test#1 identified a total of 6971 differentially expressed genes (6569 + 187 + 215, p-value < 0.05) by comparing the transcriptome of NSCs from glra4a morpholino-injected (GlyR KD) versus uninjected embryos (WT). Test#2 identified 635 differentially expressed genes between the transcriptome of NSCs from control morpholino-injected (Ctrl KD) versus uninjected (WT) (233 + 215 + 187, p-value < 0.05). 402 genes (215 + 187) were overlapping between tests #1 and #2 (in blue). Test#3 compared the transcriptome of GlyR KD versus Ctrl KD for these 402 genes and identified 187 genes for which the differential expression was significant (p-value < 0.05). These genes were compiled with 6569 genes differentially expressed in GlyR KD (in red) and compose the final list of 6756 specific genes differentially expressed in NSCs upon GlyR KD (stripped red). (B) Volcano plot showing each of the 6756 genes from the final list established above. Blue dots represent the total 6756 specific genes filtered on a p-value < 0.05. Orange dots show the 1162 genes with a /log2FC/ > 1 and red dots show the 141 genes with a /log2FC/ > 2.
Mentions: Between 65 and 89 million high quality reads per condition were sequenced and we were able to map about 86% of these reads onto the zv9 version of the zebrafish genome (ensemble release 77). Using DESeq2 algorithm, we assessed the differentially expressed genes comparing the dataset from GlyR knockdown NSCs with uninjected WT NSCs (test#1, Figure 3A) and found 6,971 differentially expressed genes (p-value < 0.05).

Bottom Line: As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis.Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down.While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Research Center of the University of Montreal Hospital Center Montréal, QC, Canada.

ABSTRACT
How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signaling that promotes calcium transients in neural stem cells (NSCs) and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signaling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine.

No MeSH data available.