Limits...
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.

Show MeSH

Related in: MedlinePlus

E2-2 alteration influences cell cycle exit of progenitors in vivo. (A)E2-2 overexpression increased cell cycle exit (EdU+Ki67–/EdU+) among the progenitor cell population (non-RGC) compared to control conditions at 2 dpe (100 ± 7.5 vs. 184.9 ± 18.0). Confocal micrographs show representative non-RGCs (GFP+) having cycled within 24 h prior to sacrifice (EdU+) and having re-engaged (Ki67+) or exited the cell cycle (Ki67–), respectively. (B) In contrast, knock-down of E2-2 decreased cell cycle exit within the progenitor pool as illustrated by the increased Ki67 immunoreactivity among EdU+ non-RGCs, when compared to control conditions (Scrambled) (100 ± 7.8 vs. 65.8 ± 1.3). (C, D)E2-2 overexpression also increased the number of Dcx+ neuroblasts (100 ± 0.5 vs. 128.1 ± 2.9) (C), whereas the number of Ascl1+ type-C cells remained unchanged (100 ± 6.4 vs. 94.6 ± 9.2) (D). (E) Percentage of cell type composition (i.e., type-B = RGC, type-C = Ascl1+, type-A = Dcx+) upon E2-2 overexpression, when compared to an empty control plasmid at 2 dpe (30.7 ± 4.6 vs. 14.2 ± 2.3, 30.0 ± 1.2 vs. 35.6 ± 2.5, 39.2 ± 0.3 vs. 50.2 ± 1.8, respectively). (F) Summary model: E2-2 orchestrates neurogenesis progression within the murine forebrain. P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (A–D). Scale bars: A &B, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4274746&req=5

Figure 5: E2-2 alteration influences cell cycle exit of progenitors in vivo. (A)E2-2 overexpression increased cell cycle exit (EdU+Ki67–/EdU+) among the progenitor cell population (non-RGC) compared to control conditions at 2 dpe (100 ± 7.5 vs. 184.9 ± 18.0). Confocal micrographs show representative non-RGCs (GFP+) having cycled within 24 h prior to sacrifice (EdU+) and having re-engaged (Ki67+) or exited the cell cycle (Ki67–), respectively. (B) In contrast, knock-down of E2-2 decreased cell cycle exit within the progenitor pool as illustrated by the increased Ki67 immunoreactivity among EdU+ non-RGCs, when compared to control conditions (Scrambled) (100 ± 7.8 vs. 65.8 ± 1.3). (C, D)E2-2 overexpression also increased the number of Dcx+ neuroblasts (100 ± 0.5 vs. 128.1 ± 2.9) (C), whereas the number of Ascl1+ type-C cells remained unchanged (100 ± 6.4 vs. 94.6 ± 9.2) (D). (E) Percentage of cell type composition (i.e., type-B = RGC, type-C = Ascl1+, type-A = Dcx+) upon E2-2 overexpression, when compared to an empty control plasmid at 2 dpe (30.7 ± 4.6 vs. 14.2 ± 2.3, 30.0 ± 1.2 vs. 35.6 ± 2.5, 39.2 ± 0.3 vs. 50.2 ± 1.8, respectively). (F) Summary model: E2-2 orchestrates neurogenesis progression within the murine forebrain. P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (A–D). Scale bars: A &B, 20 μm.

Mentions: We next tested whether the changes in proliferation observed following E-protein GoF could be explained by cell cycle exit induction. Dividing cells in S-phase that incorporated 5-ethynyl-2′-deoxyuridine (EdU) 24 h prior to sacrifice, but did not re-enter another round of cell division (i.e., cell cycle exit), would no longer express Ki67. We quantified the number of EdU+Ki67–/EdU+ cells and found a doubling in the number of progenitors that had exited the cell cycle, when E2-2 was overexpressed (Figure 5A), whereas a smaller number of progenitors exited the cell cycle upon E2-2 silencing (Figure 5B). Finally, we tested whether E2-2-induced cell cycle exit resulted in an abortive differentiation or whether cells appropriately completed their differentiation into Dcx+ neuroblasts. We observed a ~30% increase of Dcx+ neuroblasts (type-A) within the postnatal forebrain at 2 dpe (Figure 5C, Additional file 1I), which paralleled the reduction of the number of RGCs described above, whereas the number of Ascl1+ type-C cells remained unaltered (Figure 5D, Additional file 1J).


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)

E2-2 alteration influences cell cycle exit of progenitors in vivo. (A)E2-2 overexpression increased cell cycle exit (EdU+Ki67–/EdU+) among the progenitor cell population (non-RGC) compared to control conditions at 2 dpe (100 ± 7.5 vs. 184.9 ± 18.0). Confocal micrographs show representative non-RGCs (GFP+) having cycled within 24 h prior to sacrifice (EdU+) and having re-engaged (Ki67+) or exited the cell cycle (Ki67–), respectively. (B) In contrast, knock-down of E2-2 decreased cell cycle exit within the progenitor pool as illustrated by the increased Ki67 immunoreactivity among EdU+ non-RGCs, when compared to control conditions (Scrambled) (100 ± 7.8 vs. 65.8 ± 1.3). (C, D)E2-2 overexpression also increased the number of Dcx+ neuroblasts (100 ± 0.5 vs. 128.1 ± 2.9) (C), whereas the number of Ascl1+ type-C cells remained unchanged (100 ± 6.4 vs. 94.6 ± 9.2) (D). (E) Percentage of cell type composition (i.e., type-B = RGC, type-C = Ascl1+, type-A = Dcx+) upon E2-2 overexpression, when compared to an empty control plasmid at 2 dpe (30.7 ± 4.6 vs. 14.2 ± 2.3, 30.0 ± 1.2 vs. 35.6 ± 2.5, 39.2 ± 0.3 vs. 50.2 ± 1.8, respectively). (F) Summary model: E2-2 orchestrates neurogenesis progression within the murine forebrain. P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (A–D). Scale bars: A &B, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274746&req=5

Figure 5: E2-2 alteration influences cell cycle exit of progenitors in vivo. (A)E2-2 overexpression increased cell cycle exit (EdU+Ki67–/EdU+) among the progenitor cell population (non-RGC) compared to control conditions at 2 dpe (100 ± 7.5 vs. 184.9 ± 18.0). Confocal micrographs show representative non-RGCs (GFP+) having cycled within 24 h prior to sacrifice (EdU+) and having re-engaged (Ki67+) or exited the cell cycle (Ki67–), respectively. (B) In contrast, knock-down of E2-2 decreased cell cycle exit within the progenitor pool as illustrated by the increased Ki67 immunoreactivity among EdU+ non-RGCs, when compared to control conditions (Scrambled) (100 ± 7.8 vs. 65.8 ± 1.3). (C, D)E2-2 overexpression also increased the number of Dcx+ neuroblasts (100 ± 0.5 vs. 128.1 ± 2.9) (C), whereas the number of Ascl1+ type-C cells remained unchanged (100 ± 6.4 vs. 94.6 ± 9.2) (D). (E) Percentage of cell type composition (i.e., type-B = RGC, type-C = Ascl1+, type-A = Dcx+) upon E2-2 overexpression, when compared to an empty control plasmid at 2 dpe (30.7 ± 4.6 vs. 14.2 ± 2.3, 30.0 ± 1.2 vs. 35.6 ± 2.5, 39.2 ± 0.3 vs. 50.2 ± 1.8, respectively). (F) Summary model: E2-2 orchestrates neurogenesis progression within the murine forebrain. P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (A–D). Scale bars: A &B, 20 μm.
Mentions: We next tested whether the changes in proliferation observed following E-protein GoF could be explained by cell cycle exit induction. Dividing cells in S-phase that incorporated 5-ethynyl-2′-deoxyuridine (EdU) 24 h prior to sacrifice, but did not re-enter another round of cell division (i.e., cell cycle exit), would no longer express Ki67. We quantified the number of EdU+Ki67–/EdU+ cells and found a doubling in the number of progenitors that had exited the cell cycle, when E2-2 was overexpressed (Figure 5A), whereas a smaller number of progenitors exited the cell cycle upon E2-2 silencing (Figure 5B). Finally, we tested whether E2-2-induced cell cycle exit resulted in an abortive differentiation or whether cells appropriately completed their differentiation into Dcx+ neuroblasts. We observed a ~30% increase of Dcx+ neuroblasts (type-A) within the postnatal forebrain at 2 dpe (Figure 5C, Additional file 1I), which paralleled the reduction of the number of RGCs described above, whereas the number of Ascl1+ type-C cells remained unaltered (Figure 5D, Additional file 1J).

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