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E-proteins orchestrate the progression of neural stem cell differentiation in the postnatal forebrain.

Fischer B, Azim K, Hurtado-Chong A, Ramelli S, Fernández M, Raineteau O - Neural Dev (2014)

Bottom Line: Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation.Conversely, knock-down by shRNA electroporation resulted in opposite effects.Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Research Institute, ETH Zurich/University of Zurich, 8057 Zurich, Switzerland. raineteau@hifo.uzh.ch.

ABSTRACT

Background: Neural stem cell (NSC) differentiation is a complex multistep process that persists in specific regions of the postnatal forebrain and requires tight regulation throughout life. The transcriptional control of NSC proliferation and specification involves Class II (proneural) and Class V (Id1-4) basic helix-loop-helix (bHLH) proteins. In this study, we analyzed the pattern of expression of their dimerization partners, Class I bHLH proteins (E-proteins), and explored their putative role in orchestrating postnatal subventricular zone (SVZ) neurogenesis.

Results: Overexpression of a dominant-negative form of the E-protein E47 (dnE47) confirmed a crucial role for bHLH transcriptional networks in postnatal neurogenesis by dramatically blocking SVZ NSC differentiation. In situ hybridization was used in combination with RT-qPCR to measure and compare the level of expression of E-protein transcripts (E2-2, E2A, and HEB) in the neonatal and adult SVZ as well as in magnetic affinity cell sorted progenitor cells and neuroblasts. Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation. To investigate the role of E-proteins in orchestrating lineage progression, both in vitro and in vivo gain-of-function and loss-of-function experiments were performed for individual E-proteins. Overexpression of E2-2 and E2A promoted SVZ neurogenesis by enhancing not only radial glial cell differentiation but also cell cycle exit of their progeny. Conversely, knock-down by shRNA electroporation resulted in opposite effects. Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.

Conclusions: In contrast to former concepts, suggesting ubiquitous expression and subsidiary function for E-proteins to foster postnatal neurogenesis, this work unveils E-proteins as being active players in the orchestration of postnatal SVZ neurogenesis.

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E-protein mRNA expression is enriched in germinal regions of the postnatal and adult forebrain and is increased during NSC differentiation in vitro and in vivo. (A) ISH of E2-2, E2A, and HEB revealed its enhanced expression in the SVZ (dashed zone at P60), the RMS, and the dentate gyrus, when compared to non-neurogenic regions (i.e., thalamus, cortex) at P6 as well as P60. (B) Inverted greyscale quantification of A.(C)E2-2, E2A, and HEB transcripts were highly expressed in the lateral SVZ compared to the striatum (dashed zone circling both regions) (0.18 ± 0.03 vs. 0.5 ± 0.06, 0.02 ± 0.004 vs. 0.46 ± 0.08, 0.003 ± 0.0004 vs. 0.07 ± 0.01, respectively). (D) Upon differentiation, E-protein mRNA levels were upregulated in NS5 cells (two-way ANOVA followed by Dunnett’s post hoc test). Differentiation progress was confirmed by upregulation of doublecortin (Dcx) transcripts, an early neuronal marker, and normalized to GAPDH and shown as a percentage vs. D0. (E) E-protein transcription, in particular E2-2, was enhanced in more mature PSA-NCAM+ MAC sorted neuroblasts compared to earlier Prominin-1+ (or CD133+) NSCs/progenitors (100 ± 32.0 vs. 687.8 ± 226.4, 100 ± 9.9 vs. 145.4 ± 15.4, 100 ± 12.1 vs. 144.0 ± 17.4, respectively). Cell sorting efficiency was confirmed by enrichment of Dcx (100 ± 34.0 vs. 1291.3 ± 438.6) and CD24 (100 ± 17.8 vs. 270.1 ± 48.1) transcripts in neuroblasts, while the generic NSC transcripts GFAP (100 ± 20.4 vs. 22.1 ± 4.5) and Id1 (100 ± 6.5 vs. 72.8 ± 4.7) were enriched within Prominin-1+ sorted cells. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions (D, E).
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Figure 3: E-protein mRNA expression is enriched in germinal regions of the postnatal and adult forebrain and is increased during NSC differentiation in vitro and in vivo. (A) ISH of E2-2, E2A, and HEB revealed its enhanced expression in the SVZ (dashed zone at P60), the RMS, and the dentate gyrus, when compared to non-neurogenic regions (i.e., thalamus, cortex) at P6 as well as P60. (B) Inverted greyscale quantification of A.(C)E2-2, E2A, and HEB transcripts were highly expressed in the lateral SVZ compared to the striatum (dashed zone circling both regions) (0.18 ± 0.03 vs. 0.5 ± 0.06, 0.02 ± 0.004 vs. 0.46 ± 0.08, 0.003 ± 0.0004 vs. 0.07 ± 0.01, respectively). (D) Upon differentiation, E-protein mRNA levels were upregulated in NS5 cells (two-way ANOVA followed by Dunnett’s post hoc test). Differentiation progress was confirmed by upregulation of doublecortin (Dcx) transcripts, an early neuronal marker, and normalized to GAPDH and shown as a percentage vs. D0. (E) E-protein transcription, in particular E2-2, was enhanced in more mature PSA-NCAM+ MAC sorted neuroblasts compared to earlier Prominin-1+ (or CD133+) NSCs/progenitors (100 ± 32.0 vs. 687.8 ± 226.4, 100 ± 9.9 vs. 145.4 ± 15.4, 100 ± 12.1 vs. 144.0 ± 17.4, respectively). Cell sorting efficiency was confirmed by enrichment of Dcx (100 ± 34.0 vs. 1291.3 ± 438.6) and CD24 (100 ± 17.8 vs. 270.1 ± 48.1) transcripts in neuroblasts, while the generic NSC transcripts GFAP (100 ± 20.4 vs. 22.1 ± 4.5) and Id1 (100 ± 6.5 vs. 72.8 ± 4.7) were enriched within Prominin-1+ sorted cells. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions (D, E).

Mentions: Based on these findings, we further explored both spatial and temporal E-protein expression in the postnatal and adult forebrain. We first analyzed the spatial pattern of expression by in situ hybridization (ISH) at postnatal day 6 (P6) and 60 (P60). All three genes coding for E-proteins (E2-2, E2A, and HEB) were abundant in the postnatal forebrain and were enriched in germinal zones, i.e., dentate gyrus, SVZ, and RMS at P6 (Figure 3A,B). These results were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) measurement from the microdissected lateral SVZ and adjacent parenchyma (i.e., striatum). All E-protein transcripts were enriched in the SVZ, with a clear predominance of E2-2 and E2A (Figure 3C). At P60, E-protein transcripts were generally less abundant compared to earlier postnatal ages. While E2-2 expression was still enhanced in the SVZ and the RMS, E2A and HEB expression levels were only marginally higher than in non-germinal regions (Figure 3A,B), thus implying that E2-2 may be required for neuronal differentiation continually during development and into adulthood.


E-proteins orchestrate the progression of neural stem cell differentiation in the postnatal forebrain.

Fischer B, Azim K, Hurtado-Chong A, Ramelli S, Fernández M, Raineteau O - Neural Dev (2014)

E-protein mRNA expression is enriched in germinal regions of the postnatal and adult forebrain and is increased during NSC differentiation in vitro and in vivo. (A) ISH of E2-2, E2A, and HEB revealed its enhanced expression in the SVZ (dashed zone at P60), the RMS, and the dentate gyrus, when compared to non-neurogenic regions (i.e., thalamus, cortex) at P6 as well as P60. (B) Inverted greyscale quantification of A.(C)E2-2, E2A, and HEB transcripts were highly expressed in the lateral SVZ compared to the striatum (dashed zone circling both regions) (0.18 ± 0.03 vs. 0.5 ± 0.06, 0.02 ± 0.004 vs. 0.46 ± 0.08, 0.003 ± 0.0004 vs. 0.07 ± 0.01, respectively). (D) Upon differentiation, E-protein mRNA levels were upregulated in NS5 cells (two-way ANOVA followed by Dunnett’s post hoc test). Differentiation progress was confirmed by upregulation of doublecortin (Dcx) transcripts, an early neuronal marker, and normalized to GAPDH and shown as a percentage vs. D0. (E) E-protein transcription, in particular E2-2, was enhanced in more mature PSA-NCAM+ MAC sorted neuroblasts compared to earlier Prominin-1+ (or CD133+) NSCs/progenitors (100 ± 32.0 vs. 687.8 ± 226.4, 100 ± 9.9 vs. 145.4 ± 15.4, 100 ± 12.1 vs. 144.0 ± 17.4, respectively). Cell sorting efficiency was confirmed by enrichment of Dcx (100 ± 34.0 vs. 1291.3 ± 438.6) and CD24 (100 ± 17.8 vs. 270.1 ± 48.1) transcripts in neuroblasts, while the generic NSC transcripts GFAP (100 ± 20.4 vs. 22.1 ± 4.5) and Id1 (100 ± 6.5 vs. 72.8 ± 4.7) were enriched within Prominin-1+ sorted cells. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions (D, E).
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Figure 3: E-protein mRNA expression is enriched in germinal regions of the postnatal and adult forebrain and is increased during NSC differentiation in vitro and in vivo. (A) ISH of E2-2, E2A, and HEB revealed its enhanced expression in the SVZ (dashed zone at P60), the RMS, and the dentate gyrus, when compared to non-neurogenic regions (i.e., thalamus, cortex) at P6 as well as P60. (B) Inverted greyscale quantification of A.(C)E2-2, E2A, and HEB transcripts were highly expressed in the lateral SVZ compared to the striatum (dashed zone circling both regions) (0.18 ± 0.03 vs. 0.5 ± 0.06, 0.02 ± 0.004 vs. 0.46 ± 0.08, 0.003 ± 0.0004 vs. 0.07 ± 0.01, respectively). (D) Upon differentiation, E-protein mRNA levels were upregulated in NS5 cells (two-way ANOVA followed by Dunnett’s post hoc test). Differentiation progress was confirmed by upregulation of doublecortin (Dcx) transcripts, an early neuronal marker, and normalized to GAPDH and shown as a percentage vs. D0. (E) E-protein transcription, in particular E2-2, was enhanced in more mature PSA-NCAM+ MAC sorted neuroblasts compared to earlier Prominin-1+ (or CD133+) NSCs/progenitors (100 ± 32.0 vs. 687.8 ± 226.4, 100 ± 9.9 vs. 145.4 ± 15.4, 100 ± 12.1 vs. 144.0 ± 17.4, respectively). Cell sorting efficiency was confirmed by enrichment of Dcx (100 ± 34.0 vs. 1291.3 ± 438.6) and CD24 (100 ± 17.8 vs. 270.1 ± 48.1) transcripts in neuroblasts, while the generic NSC transcripts GFAP (100 ± 20.4 vs. 22.1 ± 4.5) and Id1 (100 ± 6.5 vs. 72.8 ± 4.7) were enriched within Prominin-1+ sorted cells. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions (D, E).
Mentions: Based on these findings, we further explored both spatial and temporal E-protein expression in the postnatal and adult forebrain. We first analyzed the spatial pattern of expression by in situ hybridization (ISH) at postnatal day 6 (P6) and 60 (P60). All three genes coding for E-proteins (E2-2, E2A, and HEB) were abundant in the postnatal forebrain and were enriched in germinal zones, i.e., dentate gyrus, SVZ, and RMS at P6 (Figure 3A,B). These results were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) measurement from the microdissected lateral SVZ and adjacent parenchyma (i.e., striatum). All E-protein transcripts were enriched in the SVZ, with a clear predominance of E2-2 and E2A (Figure 3C). At P60, E-protein transcripts were generally less abundant compared to earlier postnatal ages. While E2-2 expression was still enhanced in the SVZ and the RMS, E2A and HEB expression levels were only marginally higher than in non-germinal regions (Figure 3A,B), thus implying that E2-2 may be required for neuronal differentiation continually during development and into adulthood.

Bottom Line: Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation.Conversely, knock-down by shRNA electroporation resulted in opposite effects.Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Research Institute, ETH Zurich/University of Zurich, 8057 Zurich, Switzerland. raineteau@hifo.uzh.ch.

ABSTRACT

Background: Neural stem cell (NSC) differentiation is a complex multistep process that persists in specific regions of the postnatal forebrain and requires tight regulation throughout life. The transcriptional control of NSC proliferation and specification involves Class II (proneural) and Class V (Id1-4) basic helix-loop-helix (bHLH) proteins. In this study, we analyzed the pattern of expression of their dimerization partners, Class I bHLH proteins (E-proteins), and explored their putative role in orchestrating postnatal subventricular zone (SVZ) neurogenesis.

Results: Overexpression of a dominant-negative form of the E-protein E47 (dnE47) confirmed a crucial role for bHLH transcriptional networks in postnatal neurogenesis by dramatically blocking SVZ NSC differentiation. In situ hybridization was used in combination with RT-qPCR to measure and compare the level of expression of E-protein transcripts (E2-2, E2A, and HEB) in the neonatal and adult SVZ as well as in magnetic affinity cell sorted progenitor cells and neuroblasts. Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation. To investigate the role of E-proteins in orchestrating lineage progression, both in vitro and in vivo gain-of-function and loss-of-function experiments were performed for individual E-proteins. Overexpression of E2-2 and E2A promoted SVZ neurogenesis by enhancing not only radial glial cell differentiation but also cell cycle exit of their progeny. Conversely, knock-down by shRNA electroporation resulted in opposite effects. Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.

Conclusions: In contrast to former concepts, suggesting ubiquitous expression and subsidiary function for E-proteins to foster postnatal neurogenesis, this work unveils E-proteins as being active players in the orchestration of postnatal SVZ neurogenesis.

Show MeSH
Related in: MedlinePlus