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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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Related in: MedlinePlus

In vitro and in vivo modulation of bHLH function regulates neurogenesis. (A)Ascl1 nucleofection in NS5 cells caused an increased Map2 and CD24 expression whilst conversely decreasing ABCG2 mRNA expression, as detected via RT-qPCR (100 ± 19.1 vs. 299.4 ± 8.4, 100 ± 19.6 vs. 392.1 ± 46.1, 100 ± 15.2 vs. 43.8 ± 2.5, respectively). Additionally, all E-proteins (E2-2, E47, HEB) further potentiated Ascl1-induced alteration in marker expression, when co-nucleofected with Ascl1 (Map2: 299.4 ± 8.4 vs. 349 ± 21.2, 345.7 ± 10, 378.3 ± 21; CD24: 392.1 ± 46.1 vs. 423.3 ± 39.7, 508.5 ± 40.2, 426.4 ± 11.7; ABCG2: 43.8 ± 2.5 vs. 35.7 ± 2.9, 29.7 ± 0.5, 38.8 ± 0.6, respectively). (B) Schematic illustration of the dominant-negative construct of E47 (dnE47), where the nuclear localization sequence is missing, therefore preventing its nuclear translocation and transcriptional activity of its dimerizing partners (F, insert). (C) Co-nucleofection of dnE47 reduced Ascl1-induced neurogenesis as revealed by Map2 RT-qPCR measurements (100 ± 4.2 vs. 66.8 ± 8.5). (D, E) Targeted in vivo electroporation of the dnE47-RFP construct rapidly reduced RGC differentiation, as revealed by the lower proportion of non-RGCs, when compared to an empty RFP control plasmid (100 ± 5.5 vs. 12.6 ± 2.7) 2 days post-electroporation. (F) Cycling progenitors (non-RGC) were maintained proliferating (Ki67+) following dnE47 induction (100 ± 9.6 vs. 182 ± 9.6). P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: D, 20 μm.
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Figure 1: In vitro and in vivo modulation of bHLH function regulates neurogenesis. (A)Ascl1 nucleofection in NS5 cells caused an increased Map2 and CD24 expression whilst conversely decreasing ABCG2 mRNA expression, as detected via RT-qPCR (100 ± 19.1 vs. 299.4 ± 8.4, 100 ± 19.6 vs. 392.1 ± 46.1, 100 ± 15.2 vs. 43.8 ± 2.5, respectively). Additionally, all E-proteins (E2-2, E47, HEB) further potentiated Ascl1-induced alteration in marker expression, when co-nucleofected with Ascl1 (Map2: 299.4 ± 8.4 vs. 349 ± 21.2, 345.7 ± 10, 378.3 ± 21; CD24: 392.1 ± 46.1 vs. 423.3 ± 39.7, 508.5 ± 40.2, 426.4 ± 11.7; ABCG2: 43.8 ± 2.5 vs. 35.7 ± 2.9, 29.7 ± 0.5, 38.8 ± 0.6, respectively). (B) Schematic illustration of the dominant-negative construct of E47 (dnE47), where the nuclear localization sequence is missing, therefore preventing its nuclear translocation and transcriptional activity of its dimerizing partners (F, insert). (C) Co-nucleofection of dnE47 reduced Ascl1-induced neurogenesis as revealed by Map2 RT-qPCR measurements (100 ± 4.2 vs. 66.8 ± 8.5). (D, E) Targeted in vivo electroporation of the dnE47-RFP construct rapidly reduced RGC differentiation, as revealed by the lower proportion of non-RGCs, when compared to an empty RFP control plasmid (100 ± 5.5 vs. 12.6 ± 2.7) 2 days post-electroporation. (F) Cycling progenitors (non-RGC) were maintained proliferating (Ki67+) following dnE47 induction (100 ± 9.6 vs. 182 ± 9.6). P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: D, 20 μm.

Mentions: In the first instance, the role of Class I/II bHLH factors in mediating neurogenesis was assessed in vitro. The efficiency of neuronal differentiation following overexpression of Ascl1 alone or in combination with E-proteins when NS5 cells were grown in proliferative culture conditions was determined. Overexpression of Ascl1 induced a >3-fold increase in neuronal differentiation compared to an empty control plasmid, as revealed by elevated Map2 or CD24 transcript expression, both of which are immature neuron markers (Figure 1A). Cotransfection of Ascl1 with either E-protein, i.e., E2-2, E47 (E2A isoform), and HEB, further potentiated Ascl1-induced neuronal differentiation by approximately 50 to 80% when Map2 expression was measured, and to a lesser extent when CD24 transcription was probed (Figure 1A). In contrast, measurement of ABCG2, a transcript expressed in NSCs, showed opposite trends (Figure 1A). Together, these results illustrate the interchangeability of E-proteins in this cellular context and their potential to promote Ascl1-induced NS5 cell differentiation.


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)

In vitro and in vivo modulation of bHLH function regulates neurogenesis. (A)Ascl1 nucleofection in NS5 cells caused an increased Map2 and CD24 expression whilst conversely decreasing ABCG2 mRNA expression, as detected via RT-qPCR (100 ± 19.1 vs. 299.4 ± 8.4, 100 ± 19.6 vs. 392.1 ± 46.1, 100 ± 15.2 vs. 43.8 ± 2.5, respectively). Additionally, all E-proteins (E2-2, E47, HEB) further potentiated Ascl1-induced alteration in marker expression, when co-nucleofected with Ascl1 (Map2: 299.4 ± 8.4 vs. 349 ± 21.2, 345.7 ± 10, 378.3 ± 21; CD24: 392.1 ± 46.1 vs. 423.3 ± 39.7, 508.5 ± 40.2, 426.4 ± 11.7; ABCG2: 43.8 ± 2.5 vs. 35.7 ± 2.9, 29.7 ± 0.5, 38.8 ± 0.6, respectively). (B) Schematic illustration of the dominant-negative construct of E47 (dnE47), where the nuclear localization sequence is missing, therefore preventing its nuclear translocation and transcriptional activity of its dimerizing partners (F, insert). (C) Co-nucleofection of dnE47 reduced Ascl1-induced neurogenesis as revealed by Map2 RT-qPCR measurements (100 ± 4.2 vs. 66.8 ± 8.5). (D, E) Targeted in vivo electroporation of the dnE47-RFP construct rapidly reduced RGC differentiation, as revealed by the lower proportion of non-RGCs, when compared to an empty RFP control plasmid (100 ± 5.5 vs. 12.6 ± 2.7) 2 days post-electroporation. (F) Cycling progenitors (non-RGC) were maintained proliferating (Ki67+) following dnE47 induction (100 ± 9.6 vs. 182 ± 9.6). P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: D, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: In vitro and in vivo modulation of bHLH function regulates neurogenesis. (A)Ascl1 nucleofection in NS5 cells caused an increased Map2 and CD24 expression whilst conversely decreasing ABCG2 mRNA expression, as detected via RT-qPCR (100 ± 19.1 vs. 299.4 ± 8.4, 100 ± 19.6 vs. 392.1 ± 46.1, 100 ± 15.2 vs. 43.8 ± 2.5, respectively). Additionally, all E-proteins (E2-2, E47, HEB) further potentiated Ascl1-induced alteration in marker expression, when co-nucleofected with Ascl1 (Map2: 299.4 ± 8.4 vs. 349 ± 21.2, 345.7 ± 10, 378.3 ± 21; CD24: 392.1 ± 46.1 vs. 423.3 ± 39.7, 508.5 ± 40.2, 426.4 ± 11.7; ABCG2: 43.8 ± 2.5 vs. 35.7 ± 2.9, 29.7 ± 0.5, 38.8 ± 0.6, respectively). (B) Schematic illustration of the dominant-negative construct of E47 (dnE47), where the nuclear localization sequence is missing, therefore preventing its nuclear translocation and transcriptional activity of its dimerizing partners (F, insert). (C) Co-nucleofection of dnE47 reduced Ascl1-induced neurogenesis as revealed by Map2 RT-qPCR measurements (100 ± 4.2 vs. 66.8 ± 8.5). (D, E) Targeted in vivo electroporation of the dnE47-RFP construct rapidly reduced RGC differentiation, as revealed by the lower proportion of non-RGCs, when compared to an empty RFP control plasmid (100 ± 5.5 vs. 12.6 ± 2.7) 2 days post-electroporation. (F) Cycling progenitors (non-RGC) were maintained proliferating (Ki67+) following dnE47 induction (100 ± 9.6 vs. 182 ± 9.6). P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: D, 20 μm.
Mentions: In the first instance, the role of Class I/II bHLH factors in mediating neurogenesis was assessed in vitro. The efficiency of neuronal differentiation following overexpression of Ascl1 alone or in combination with E-proteins when NS5 cells were grown in proliferative culture conditions was determined. Overexpression of Ascl1 induced a >3-fold increase in neuronal differentiation compared to an empty control plasmid, as revealed by elevated Map2 or CD24 transcript expression, both of which are immature neuron markers (Figure 1A). Cotransfection of Ascl1 with either E-protein, i.e., E2-2, E47 (E2A isoform), and HEB, further potentiated Ascl1-induced neuronal differentiation by approximately 50 to 80% when Map2 expression was measured, and to a lesser extent when CD24 transcription was probed (Figure 1A). In contrast, measurement of ABCG2, a transcript expressed in NSCs, showed opposite trends (Figure 1A). Together, these results illustrate the interchangeability of E-proteins in this cellular context and their potential to promote Ascl1-induced NS5 cell differentiation.

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