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Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis

Nielsen, Joseph A, Lau, Pierre, Maric, Dragan, Barker, Jeffery L, Hudson, Lynn D·2009·BMC Neuroscience
DOI 10.1186/1471-2202-10-98PMC PMC2736963
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Figure 1 Neuronal progenitors can be purified directly from the rat telencephalon using flow cytometry. Dissociates were labeled using the pan-neuronal markers TnTx and ChTx in addition to A2B5 and Jones, which identify a separate lineage that also includes neuronal progenitors and later neuroglial progenitors (NGP). A. Bivariate FACS plots reveal a developmental increase in TnTx+/ChTx+/A2B5-/Jones- early neuronal progenitors (ENP) from 3% to 20.5% and the emergence of late neuronal progenitors (LNP) at E13. TnTx+/ChTx+ immunofluorescence signals were used to sort-purify populations of viable cells at embryonic days E11, E12 and E13. Sorting gates were set to collect TnTx+/ChTx+/A2B5-/Jones- ENPs and, at E13, LNPs, which were identified on the basis of relative fluorescence intensities of TnTx and ChTx staining reactions (upper left quadrant). B. Analysis of total RNA extracted from NPs isolated by FACS shows the presence of intact 18S and 28S rRNA bands (arrow heads). C. Principle component analysis demonstrates the reproducibility of the biological replicates. Red circles = E11 ENPs; yellow circles = E12 ENPs; light blue circles = E13 ENPs; dark blue circles = E13 LNPs. D. Scatter plot of two E11 ENP biological replicates. Correlation analysis was performed on the biological replicates within each group. The correlation coefficient was greater than 0.94 for each replicate.
Figure 1 Neuronal progenitors can be purified directly from the rat telencephalon using flow cytometry. Dissociates were labeled using the pan-neuronal markers TnTx and ChTx in addition to A2B5 and Jones, which identify a separate lineage that also includes neuronal progenitors and later neuroglial progenitors (NGP). A. Bivariate FACS plots reveal a developmental increase in TnTx+/ChTx+/A2B5-/Jones- early neuronal progenitors (ENP) from 3% to 20.5% and the emergence of late neuronal progenitors (LNP) at E13. TnTx+/ChTx+ immunofluorescence signals were used to sort-purify populations of viable cells at embryonic days E11, E12 and E13. Sorting gates were set to collect TnTx+/ChTx+/A2B5-/Jones- ENPs and, at E13, LNPs, which were identified on the basis of relative fluorescence intensities of TnTx and ChTx staining reactions (upper left quadrant). B. Analysis of total RNA extracted from NPs isolated by FACS shows the presence of intact 18S and 28S rRNA bands (arrow heads). C. Principle component analysis demonstrates the reproducibility of the biological replicates. Red circles = E11 ENPs; yellow circles = E12 ENPs; light blue circles = E13 ENPs; dark blue circles = E13 LNPs. D. Scatter plot of two E11 ENP biological replicates. Correlation analysis was performed on the biological replicates within each group. The correlation coefficient was greater than 0.94 for each replicate.CC BY · PubMed Central
Figure 2 Neuronal progenitors are located in the ventral telencephalon near the pial surface. Sagittal sections of E12 rat ventral telencephalon were immunostained with anti-nestin (blue), anti-PCNA (yellow), anti-CDw60/Jones (green) and anti-beta-tubulin III/Tuj1 (red). Tuj1 antibody was used in place of TnTx to provide a better visualization of neuronal cell bodies. Insets 1–3 in the top panel are enlarged in the bottom panels (1A-D, 2A-D, 3A-D) to show the anatomical distribution of the 4 major neural phenotypes, which were classified as follows: (a) Nestin+PCNA+Jones-Tuj1- neural stem/precursor cells, (b) Nestin+PCNA+Jones-Tuj1+ neuronal progenitors and post-mitotic neurons, which are predominantly Nestin-PCNA-, (c) Nestin+PCNA+Jones+Tuj1- neuroglial progenitors and (d) Nestin+PCNA+Jones+Tuj1+ neuronal progenitors, The majority of Jones-TnTx+ neuronal progenitors isolated by FACS for microarray analysis in this study are represented by Jones-Tuj1+ cells (population b) located near the pial surface as shown in panels 3C and 3D.
Figure 2 Neuronal progenitors are located in the ventral telencephalon near the pial surface. Sagittal sections of E12 rat ventral telencephalon were immunostained with anti-nestin (blue), anti-PCNA (yellow), anti-CDw60/Jones (green) and anti-beta-tubulin III/Tuj1 (red). Tuj1 antibody was used in place of TnTx to provide a better visualization of neuronal cell bodies. Insets 1–3 in the top panel are enlarged in the bottom panels (1A-D, 2A-D, 3A-D) to show the anatomical distribution of the 4 major neural phenotypes, which were classified as follows: (a) Nestin+PCNA+Jones-Tuj1- neural stem/precursor cells, (b) Nestin+PCNA+Jones-Tuj1+ neuronal progenitors and post-mitotic neurons, which are predominantly Nestin-PCNA-, (c) Nestin+PCNA+Jones+Tuj1- neuroglial progenitors and (d) Nestin+PCNA+Jones+Tuj1+ neuronal progenitors, The majority of Jones-TnTx+ neuronal progenitors isolated by FACS for microarray analysis in this study are represented by Jones-Tuj1+ cells (population b) located near the pial surface as shown in panels 3C and 3D.CC BY · PubMed Central
Figure 3 mRNAs and miRNAs are dynamically regulated during the onset of cortical neurogenesis. K-means clustering analysis was applied to the normalized expression data (see Materials and Methods). Two major classes of differentially regulated mRNAs (A, B) and miRNAs (C, D) were identified. The mRNA and miRNA expression levels were colored by the relative expression levels in E11 ENPs with red being the highest and blue being the lowest globally normalized expression level.
Figure 3 mRNAs and miRNAs are dynamically regulated during the onset of cortical neurogenesis. K-means clustering analysis was applied to the normalized expression data (see Materials and Methods). Two major classes of differentially regulated mRNAs (A, B) and miRNAs (C, D) were identified. The mRNA and miRNA expression levels were colored by the relative expression levels in E11 ENPs with red being the highest and blue being the lowest globally normalized expression level.CC BY · PubMed Central
Figure 4 Differentially expressed mRNAs detected by microarrays were validated with qRT-PCR and immunohistochemistry. A. The cycle threshold of Klf7 is shifted to the left in E13 ENPs confirming an increase in Klf7 mRNA expression level, while the beta-actin mRNA expression level remains nearly constant. B. The fold changes from E11 to E13 for seven transcription factors identified by microarrays (black bars) and qRT-PCR (grey bars) are plotted. C, D. Cutl1 protein (red fluorescence) is expressed at E13 in the differentiating zone (DZ) of the ventral (C) and dorsal (D) telencephalon. The blue fluorescence depicts nuclear counterstain with DAPI. The lateral ventricle (LV) is at bottom of the figure. NE = neuroepithelium. Scale bar = 20 microns.
Figure 4 Differentially expressed mRNAs detected by microarrays were validated with qRT-PCR and immunohistochemistry. A. The cycle threshold of Klf7 is shifted to the left in E13 ENPs confirming an increase in Klf7 mRNA expression level, while the beta-actin mRNA expression level remains nearly constant. B. The fold changes from E11 to E13 for seven transcription factors identified by microarrays (black bars) and qRT-PCR (grey bars) are plotted. C, D. Cutl1 protein (red fluorescence) is expressed at E13 in the differentiating zone (DZ) of the ventral (C) and dorsal (D) telencephalon. The blue fluorescence depicts nuclear counterstain with DAPI. The lateral ventricle (LV) is at bottom of the figure. NE = neuroepithelium. Scale bar = 20 microns.CC BY · PubMed Central
Figure 5 Gene ontology (GO) analysis of differentially regulated transcripts. The mRNAs encoding transcription factors make up the second largest class of mRNAs being down-regulated (A) and the largest class of mRNAs being up-regulated (B). Differentially regulated gene lists (see methods) were imported into DAVID for GO analysis. The analysis was confined to the biological process category (GO level 5). The 15 GO categories with the highest number of mRNAs annotated were included with the number of mRNAs in each category graphed on the x-axis.
Figure 5 Gene ontology (GO) analysis of differentially regulated transcripts. The mRNAs encoding transcription factors make up the second largest class of mRNAs being down-regulated (A) and the largest class of mRNAs being up-regulated (B). Differentially regulated gene lists (see methods) were imported into DAVID for GO analysis. The analysis was confined to the biological process category (GO level 5). The 15 GO categories with the highest number of mRNAs annotated were included with the number of mRNAs in each category graphed on the x-axis.CC BY · PubMed Central
Figure 6 miRNA expression is negatively correlated with target mRNA expression. Predicted target mRNAs for each miRNA were identified using TargetScan 4.0 and compared with the lists of experimentally-determined, differentially-regulated mRNAs. A two-tailed Fisher's Exact Test was used to determine whether there were more predicted target mRNAs with differential expression than would be expected by chance (p < 0.05, above heavy black line at 4.3). The negative log of the p-value is plotted on the x-axis for both down-regulated mRNAs (grey) and up-regulated mRNAs (black).
Figure 6 miRNA expression is negatively correlated with target mRNA expression. Predicted target mRNAs for each miRNA were identified using TargetScan 4.0 and compared with the lists of experimentally-determined, differentially-regulated mRNAs. A two-tailed Fisher's Exact Test was used to determine whether there were more predicted target mRNAs with differential expression than would be expected by chance (p < 0.05, above heavy black line at 4.3). The negative log of the p-value is plotted on the x-axis for both down-regulated mRNAs (grey) and up-regulated mRNAs (black).CC BY · PubMed Central
Figure 7 GO analysis identifies potential mRNA functional categories regulated by miRNAs during neuron differentiation. GO analysis was performed on mRNAs predicted to be targets of miRNAs, which were negatively correlated with target mRNA expression. The GO analysis was confined to the biological process category (GO level 5). Only categories with a p-value greater that 0.05 are included. The negative log of the p-value is plotted on the x-axis.
Figure 7 GO analysis identifies potential mRNA functional categories regulated by miRNAs during neuron differentiation. GO analysis was performed on mRNAs predicted to be targets of miRNAs, which were negatively correlated with target mRNA expression. The GO analysis was confined to the biological process category (GO level 5). Only categories with a p-value greater that 0.05 are included. The negative log of the p-value is plotted on the x-axis.CC BY · PubMed Central
Figure 8 Network analysis identifies miRNAs that are predicted to target multiple transcription factors. The small grey circles identify TFs that were up-regulated in neuronal progenitors between E11 and E13. Larger black circles identify miRNAs that were down-regulated in neuronal progenitors between E11 and E13. The connection lines indicate that a miRNA binding site was identified in the 3' UTR of the transcription factor using TargetScan for miRNA target prediction.
Figure 8 Network analysis identifies miRNAs that are predicted to target multiple transcription factors. The small grey circles identify TFs that were up-regulated in neuronal progenitors between E11 and E13. Larger black circles identify miRNAs that were down-regulated in neuronal progenitors between E11 and E13. The connection lines indicate that a miRNA binding site was identified in the 3' UTR of the transcription factor using TargetScan for miRNA target prediction.CC BY · PubMed Central

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Authors
Nielsen, J. A., Lau, P., Maric, D., Barker, J. L., & Hudson, L. D.
Venue
BMC Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2009-08-19
Volume
10
Issue
1
ISSN
1471-2202
DOI
10.1186/1471-2202-10-98
PMC
PMC2736963
Cited by
66
References
2
Language
en
Licence
creativecommons.org/licenses/by/2.0 (tdm)

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