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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.


Related in: MedlinePlus

Glycine receptor (GlyRs) knock-down NSCs. (A) Experimental protocol to purify NSCs from (i) uninjected, (ii) Ctrl MO-injected, and (iii) glra4a MO-injected. gfap:GFP embryos were injected at the one-cell stage and let develop until 20hpf. NSCs were sorted by FACS and harvested for total RNA extraction. (B)glra4a morpholino targets 24 nucleotides (in red) downstream to the transcription starting site. A 100 bp fragment encompassing the end of the 5UTR and the 5 part of the first exon was cloned in frame with eGFP coding sequence downstream to the CMV promoter. (C) Embryos co-injected with the glra4a-eGFP construct and Ctrl morpholino depict broad GFP fluorescence at 20 hpf (upper right). However, co-injection with glra4a morpholino abolishes fluorescence (lower right) thus validating the use of both Ctrl and glra4a morpholinos.
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Figure 2: Glycine receptor (GlyRs) knock-down NSCs. (A) Experimental protocol to purify NSCs from (i) uninjected, (ii) Ctrl MO-injected, and (iii) glra4a MO-injected. gfap:GFP embryos were injected at the one-cell stage and let develop until 20hpf. NSCs were sorted by FACS and harvested for total RNA extraction. (B)glra4a morpholino targets 24 nucleotides (in red) downstream to the transcription starting site. A 100 bp fragment encompassing the end of the 5UTR and the 5 part of the first exon was cloned in frame with eGFP coding sequence downstream to the CMV promoter. (C) Embryos co-injected with the glra4a-eGFP construct and Ctrl morpholino depict broad GFP fluorescence at 20 hpf (upper right). However, co-injection with glra4a morpholino abolishes fluorescence (lower right) thus validating the use of both Ctrl and glra4a morpholinos.

Mentions: We used the same procedures to purify NSCs from (i) uninjected 20 hpf embryos, (ii) embryos injected with a 5 base-mismatch control morpholino, and (iii) embryos knocked-down for GlyR using a specific glra4a morpholino (Figure 2A). To validate the in vivo efficacy and selectivity of our morpholino, we cloned a 0.1 kb fragment of the glra4a region containing the morpholino target site (in red in Figure 2B) in frame with the coding sequence of GFP. After co-injection of this construct and our control morpholino, 92% of the embryos (n = 49/53) depicted broad GFP fluorescence, thus validating the expression of the fusion protein (Figure 2C). However, when co-injected with glra4a morpholino, GFP was undetectable in 36 out of 38 of the injected embryos (95%). These results validate the selectivity of our glra4a morpholino compared to the 5 base-mismatch control morpholino. For each condition (uninjected, control, and GlyR knockdown), we dissociated between 60 and 80 gfap:GFP+ embryos from which we successfully FACS-purified between 5.19 and 9.75 × 105 GFP+ cells (Figure 2A). Each treatment condition was processed in duplicate from independent batches of eggs and total RNA was extracted reaching a RIN higher than 8.50.


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)

Glycine receptor (GlyRs) knock-down NSCs. (A) Experimental protocol to purify NSCs from (i) uninjected, (ii) Ctrl MO-injected, and (iii) glra4a MO-injected. gfap:GFP embryos were injected at the one-cell stage and let develop until 20hpf. NSCs were sorted by FACS and harvested for total RNA extraction. (B)glra4a morpholino targets 24 nucleotides (in red) downstream to the transcription starting site. A 100 bp fragment encompassing the end of the 5UTR and the 5 part of the first exon was cloned in frame with eGFP coding sequence downstream to the CMV promoter. (C) Embryos co-injected with the glra4a-eGFP construct and Ctrl morpholino depict broad GFP fluorescence at 20 hpf (upper right). However, co-injection with glra4a morpholino abolishes fluorescence (lower right) thus validating the use of both Ctrl and glra4a morpholinos.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Glycine receptor (GlyRs) knock-down NSCs. (A) Experimental protocol to purify NSCs from (i) uninjected, (ii) Ctrl MO-injected, and (iii) glra4a MO-injected. gfap:GFP embryos were injected at the one-cell stage and let develop until 20hpf. NSCs were sorted by FACS and harvested for total RNA extraction. (B)glra4a morpholino targets 24 nucleotides (in red) downstream to the transcription starting site. A 100 bp fragment encompassing the end of the 5UTR and the 5 part of the first exon was cloned in frame with eGFP coding sequence downstream to the CMV promoter. (C) Embryos co-injected with the glra4a-eGFP construct and Ctrl morpholino depict broad GFP fluorescence at 20 hpf (upper right). However, co-injection with glra4a morpholino abolishes fluorescence (lower right) thus validating the use of both Ctrl and glra4a morpholinos.
Mentions: We used the same procedures to purify NSCs from (i) uninjected 20 hpf embryos, (ii) embryos injected with a 5 base-mismatch control morpholino, and (iii) embryos knocked-down for GlyR using a specific glra4a morpholino (Figure 2A). To validate the in vivo efficacy and selectivity of our morpholino, we cloned a 0.1 kb fragment of the glra4a region containing the morpholino target site (in red in Figure 2B) in frame with the coding sequence of GFP. After co-injection of this construct and our control morpholino, 92% of the embryos (n = 49/53) depicted broad GFP fluorescence, thus validating the expression of the fusion protein (Figure 2C). However, when co-injected with glra4a morpholino, GFP was undetectable in 36 out of 38 of the injected embryos (95%). These results validate the selectivity of our glra4a morpholino compared to the 5 base-mismatch control morpholino. For each condition (uninjected, control, and GlyR knockdown), we dissociated between 60 and 80 gfap:GFP+ embryos from which we successfully FACS-purified between 5.19 and 9.75 × 105 GFP+ cells (Figure 2A). Each treatment condition was processed in duplicate from independent batches of eggs and total RNA was extracted reaching a RIN higher than 8.50.

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.


Related in: MedlinePlus