Limits...
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.


Identified signaling pathways downstream to glycine in NSCs. Representation of the pathways identified and of the key genes involved according to KEGG and IPA. For each signaling pathway (A) calcium, (B) TGF-beta, (C) SHH, (D) WNT, and (E) P53, upregulated genes upon GlyR KD are noted in green and downregulated genes are in red and underlined. Genes highlighted in yellow have been tested by RT-qPCR to confirm RNA-sequencing data (see Figure 5).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4815022&req=5

Figure 4: Identified signaling pathways downstream to glycine in NSCs. Representation of the pathways identified and of the key genes involved according to KEGG and IPA. For each signaling pathway (A) calcium, (B) TGF-beta, (C) SHH, (D) WNT, and (E) P53, upregulated genes upon GlyR KD are noted in green and downregulated genes are in red and underlined. Genes highlighted in yellow have been tested by RT-qPCR to confirm RNA-sequencing data (see Figure 5).

Mentions: Using the freely available DAVID bioinformatics resources (Huang da et al., 2009) and through orthologous extrapolation from the IPA software (Ingenuity Pathway Analysis, QIAGEN) we performed pathway analysis using our list of 1,162 specific genes differentially expressed in NSCs upon GlyR knockdown. To this aim, we used the KEGG annotation of identified pathways involving genes of our list. Interestingly, five main molecular pathways came out from this analysis as being enriched in our dataset (Figures 4A–E): signaling by (i) calcium, (ii) TGF-beta, (iii) sonic hedgehog, (iv) Wnt, and (v) p53-related apoptosis. Regarding calcium signaling, we found CACNA2D4B and other calcium channels (CACNG1, CACNG8B, and CACNB2B) to be upregulated. Consistently, upon GlyR knockdown, the ITPR1 intracellular calcium channel expressed at the endoplasmic reticulum was also upregulated. Besides, the expression of genes encoding the Gαq GNA14 and the downstream phospholipases C (PLCD1A/4A), which activate ITRP1, were also upregulated in our dataset. Interestingly, we found that some calcium-dependent proteins such as calmodulin kinases CAMK2D1 and CAMK4, the proline-rich tyrosine kinase PTK2BB and the nitric oxide synthase 1 NOS1 were upregulated in NSCs upon GlyR knockdown. Finally, some transcription factors regulated by calmodulin kinases and belonging to the CREB complex were downregulated in our dataset (ATF4B2, ATF7IP, and ATF7A) (Figure 4A).


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)

Identified signaling pathways downstream to glycine in NSCs. Representation of the pathways identified and of the key genes involved according to KEGG and IPA. For each signaling pathway (A) calcium, (B) TGF-beta, (C) SHH, (D) WNT, and (E) P53, upregulated genes upon GlyR KD are noted in green and downregulated genes are in red and underlined. Genes highlighted in yellow have been tested by RT-qPCR to confirm RNA-sequencing data (see Figure 5).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4815022&req=5

Figure 4: Identified signaling pathways downstream to glycine in NSCs. Representation of the pathways identified and of the key genes involved according to KEGG and IPA. For each signaling pathway (A) calcium, (B) TGF-beta, (C) SHH, (D) WNT, and (E) P53, upregulated genes upon GlyR KD are noted in green and downregulated genes are in red and underlined. Genes highlighted in yellow have been tested by RT-qPCR to confirm RNA-sequencing data (see Figure 5).
Mentions: Using the freely available DAVID bioinformatics resources (Huang da et al., 2009) and through orthologous extrapolation from the IPA software (Ingenuity Pathway Analysis, QIAGEN) we performed pathway analysis using our list of 1,162 specific genes differentially expressed in NSCs upon GlyR knockdown. To this aim, we used the KEGG annotation of identified pathways involving genes of our list. Interestingly, five main molecular pathways came out from this analysis as being enriched in our dataset (Figures 4A–E): signaling by (i) calcium, (ii) TGF-beta, (iii) sonic hedgehog, (iv) Wnt, and (v) p53-related apoptosis. Regarding calcium signaling, we found CACNA2D4B and other calcium channels (CACNG1, CACNG8B, and CACNB2B) to be upregulated. Consistently, upon GlyR knockdown, the ITPR1 intracellular calcium channel expressed at the endoplasmic reticulum was also upregulated. Besides, the expression of genes encoding the Gαq GNA14 and the downstream phospholipases C (PLCD1A/4A), which activate ITRP1, were also upregulated in our dataset. Interestingly, we found that some calcium-dependent proteins such as calmodulin kinases CAMK2D1 and CAMK4, the proline-rich tyrosine kinase PTK2BB and the nitric oxide synthase 1 NOS1 were upregulated in NSCs upon GlyR knockdown. Finally, some transcription factors regulated by calmodulin kinases and belonging to the CREB complex were downregulated in our dataset (ATF4B2, ATF7IP, and ATF7A) (Figure 4A).

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.