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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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E47 fails to trigger Ascl1 to switch from proliferation to differentiation target genes. (A)Map2 mRNA expression was elevated when Ascl1 was overexpressed in isolated neurospheres in vitro (100 ± 19.1 vs. 299.4 ± 5.8). Concomitant knock-down of E2-2 or E2A using shRNA, respectively, reduced or abolished Ascl1-dependent Map2 expression (299.4 ± 5.8 vs. 222.2 ± 3.7 or 94.9 ± 9.8, respectively). More strikingly, Ascl1 silencing decreased Map2 expression level to control conditions, when E2-2 (100 ± 19.1 vs. 242.4 ± 34.2, 242.4 ± 34.2 vs. 100.3 ± 4.9) or E47 (100 ± 19.1 vs. 214.6 ± 7.1, 214.6 ± 7.1 vs. 125.7 ± 13.6) was overexpressed. (B) Although significant expression changes could be detected, E-protein gain-of-function did not systematically promote activation of Ascl1 target genes mediating cell cycle arrest. Inversely, E-protein silencing did not activate Ascl1 target genes mediating cell cycle progression. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions.
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Figure 6: E47 fails to trigger Ascl1 to switch from proliferation to differentiation target genes. (A)Map2 mRNA expression was elevated when Ascl1 was overexpressed in isolated neurospheres in vitro (100 ± 19.1 vs. 299.4 ± 5.8). Concomitant knock-down of E2-2 or E2A using shRNA, respectively, reduced or abolished Ascl1-dependent Map2 expression (299.4 ± 5.8 vs. 222.2 ± 3.7 or 94.9 ± 9.8, respectively). More strikingly, Ascl1 silencing decreased Map2 expression level to control conditions, when E2-2 (100 ± 19.1 vs. 242.4 ± 34.2, 242.4 ± 34.2 vs. 100.3 ± 4.9) or E47 (100 ± 19.1 vs. 214.6 ± 7.1, 214.6 ± 7.1 vs. 125.7 ± 13.6) was overexpressed. (B) Although significant expression changes could be detected, E-protein gain-of-function did not systematically promote activation of Ascl1 target genes mediating cell cycle arrest. Inversely, E-protein silencing did not activate Ascl1 target genes mediating cell cycle progression. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions.

Mentions: E-proteins induce NSC differentiation by transactivation of bHLH proneural proteins. Among those is Ascl1, expressed mainly by progenitor cells in the postnatal forebrain [19,38]. We confirmed the interdependence of E-proteins and Ascl1 by NSC differentiation induction, combining GoF and LoF experiments in isolated progenitors in vitro. Ascl1 knock-down resulted in a complete loss of Map2 mRNA transcription, when E2-2 or E47 were concomitantly overexpressed. This confirms that E-protein activity is largely dependent on Ascl1 expression. On the other hand, Ascl1 is still able to exert its function although to a lesser extent upon silencing of E2-2, but not after silencing E2A, illustrating both redundancy but also a strong dependency on E2A expression in SVZ-derived progenitor cells (Figure 6A).


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)

E47 fails to trigger Ascl1 to switch from proliferation to differentiation target genes. (A)Map2 mRNA expression was elevated when Ascl1 was overexpressed in isolated neurospheres in vitro (100 ± 19.1 vs. 299.4 ± 5.8). Concomitant knock-down of E2-2 or E2A using shRNA, respectively, reduced or abolished Ascl1-dependent Map2 expression (299.4 ± 5.8 vs. 222.2 ± 3.7 or 94.9 ± 9.8, respectively). More strikingly, Ascl1 silencing decreased Map2 expression level to control conditions, when E2-2 (100 ± 19.1 vs. 242.4 ± 34.2, 242.4 ± 34.2 vs. 100.3 ± 4.9) or E47 (100 ± 19.1 vs. 214.6 ± 7.1, 214.6 ± 7.1 vs. 125.7 ± 13.6) was overexpressed. (B) Although significant expression changes could be detected, E-protein gain-of-function did not systematically promote activation of Ascl1 target genes mediating cell cycle arrest. Inversely, E-protein silencing did not activate Ascl1 target genes mediating cell cycle progression. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions.
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Related In: Results  -  Collection

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Figure 6: E47 fails to trigger Ascl1 to switch from proliferation to differentiation target genes. (A)Map2 mRNA expression was elevated when Ascl1 was overexpressed in isolated neurospheres in vitro (100 ± 19.1 vs. 299.4 ± 5.8). Concomitant knock-down of E2-2 or E2A using shRNA, respectively, reduced or abolished Ascl1-dependent Map2 expression (299.4 ± 5.8 vs. 222.2 ± 3.7 or 94.9 ± 9.8, respectively). More strikingly, Ascl1 silencing decreased Map2 expression level to control conditions, when E2-2 (100 ± 19.1 vs. 242.4 ± 34.2, 242.4 ± 34.2 vs. 100.3 ± 4.9) or E47 (100 ± 19.1 vs. 214.6 ± 7.1, 214.6 ± 7.1 vs. 125.7 ± 13.6) was overexpressed. (B) Although significant expression changes could be detected, E-protein gain-of-function did not systematically promote activation of Ascl1 target genes mediating cell cycle arrest. Inversely, E-protein silencing did not activate Ascl1 target genes mediating cell cycle progression. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions.
Mentions: E-proteins induce NSC differentiation by transactivation of bHLH proneural proteins. Among those is Ascl1, expressed mainly by progenitor cells in the postnatal forebrain [19,38]. We confirmed the interdependence of E-proteins and Ascl1 by NSC differentiation induction, combining GoF and LoF experiments in isolated progenitors in vitro. Ascl1 knock-down resulted in a complete loss of Map2 mRNA transcription, when E2-2 or E47 were concomitantly overexpressed. This confirms that E-protein activity is largely dependent on Ascl1 expression. On the other hand, Ascl1 is still able to exert its function although to a lesser extent upon silencing of E2-2, but not after silencing E2A, illustrating both redundancy but also a strong dependency on E2A expression in SVZ-derived progenitor cells (Figure 6A).

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