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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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E2-2 and E2A sequentially affect RGC differentiation and progenitor proliferation. (A) Representative illustration of a green fluorescence protein (GFP) plasmid electroporation into the lateral wall of the LV. (B)In vitro characterization of the shRNAs used in this study. shRNA against E2-2 or E2A decreased E2-2 or E2A expression (100 ± 18.9 vs. 17.2 ± 2.4 or 100 ± 20.2 vs. 20.9 ± 2.5, respectively) within neurosphere cultures, when co-nucleofected with E2-2 or E47 overexpression plasmids, respectively. (C)E2-2 and E47 overexpression constructs increased transition from RGCs (bipolar processes, apical connection to LV lumen) to non-RGCs (no processes, spherical morphologies, distant from the LV wall) at 2 dpe (100 ± 3.9 vs. 130.5 ± 3.0 or 139.7 ± 1.4, respectively). When shRNA plasmids against E2-2 or E2A were applied, most electroporated cells maintained a RGC morphology, as reflected by the reduced ratio of non-RGC cells compared to control conditions (100 ± 3 vs. 81 ± 6.4 or 82.9 ± 3.6, respectively). Confocal micrographs illustrate RGCs and non-RGCs coexisting in the neurogenic niche. Inserts show a schematic view of the RGC to non-RGC transition. (D)E2-2 and E47 overexpression constructs decreased Ki67 immunoreactivity within progenitors (non-RGC) at 2 dpe (100 ± 5.6 vs. 61 ± 3.5 or 64.7 ± 4.1, respectively). shRNA constructs against E2-2 and E2A-induced opposite effects (100 ± 8 vs. 216 ± 13.2 or 161.9 ± 12.7, respectively). Confocal micrographs illustrate representative non-RGCs positive or negative for Ki67, respectively. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: C, 20 μm; D, 10 μm.
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Figure 4: E2-2 and E2A sequentially affect RGC differentiation and progenitor proliferation. (A) Representative illustration of a green fluorescence protein (GFP) plasmid electroporation into the lateral wall of the LV. (B)In vitro characterization of the shRNAs used in this study. shRNA against E2-2 or E2A decreased E2-2 or E2A expression (100 ± 18.9 vs. 17.2 ± 2.4 or 100 ± 20.2 vs. 20.9 ± 2.5, respectively) within neurosphere cultures, when co-nucleofected with E2-2 or E47 overexpression plasmids, respectively. (C)E2-2 and E47 overexpression constructs increased transition from RGCs (bipolar processes, apical connection to LV lumen) to non-RGCs (no processes, spherical morphologies, distant from the LV wall) at 2 dpe (100 ± 3.9 vs. 130.5 ± 3.0 or 139.7 ± 1.4, respectively). When shRNA plasmids against E2-2 or E2A were applied, most electroporated cells maintained a RGC morphology, as reflected by the reduced ratio of non-RGC cells compared to control conditions (100 ± 3 vs. 81 ± 6.4 or 82.9 ± 3.6, respectively). Confocal micrographs illustrate RGCs and non-RGCs coexisting in the neurogenic niche. Inserts show a schematic view of the RGC to non-RGC transition. (D)E2-2 and E47 overexpression constructs decreased Ki67 immunoreactivity within progenitors (non-RGC) at 2 dpe (100 ± 5.6 vs. 61 ± 3.5 or 64.7 ± 4.1, respectively). shRNA constructs against E2-2 and E2A-induced opposite effects (100 ± 8 vs. 216 ± 13.2 or 161.9 ± 12.7, respectively). Confocal micrographs illustrate representative non-RGCs positive or negative for Ki67, respectively. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: C, 20 μm; D, 10 μm.

Mentions: We next performed further E-protein gain-of-function (GoF) and loss-of-function (LoF) experiments in the postnatal lateral SVZ, by means of plasmid electroporation (as above) (Figure 4A). We focused this functional characterization onto E2-2, which appears to be the most prominent E-protein expressed in the postnatal and adult SVZ (see above), and E2A, which is the best studied E-protein in CNS development [23,25,29,35-37].


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 and E2A sequentially affect RGC differentiation and progenitor proliferation. (A) Representative illustration of a green fluorescence protein (GFP) plasmid electroporation into the lateral wall of the LV. (B)In vitro characterization of the shRNAs used in this study. shRNA against E2-2 or E2A decreased E2-2 or E2A expression (100 ± 18.9 vs. 17.2 ± 2.4 or 100 ± 20.2 vs. 20.9 ± 2.5, respectively) within neurosphere cultures, when co-nucleofected with E2-2 or E47 overexpression plasmids, respectively. (C)E2-2 and E47 overexpression constructs increased transition from RGCs (bipolar processes, apical connection to LV lumen) to non-RGCs (no processes, spherical morphologies, distant from the LV wall) at 2 dpe (100 ± 3.9 vs. 130.5 ± 3.0 or 139.7 ± 1.4, respectively). When shRNA plasmids against E2-2 or E2A were applied, most electroporated cells maintained a RGC morphology, as reflected by the reduced ratio of non-RGC cells compared to control conditions (100 ± 3 vs. 81 ± 6.4 or 82.9 ± 3.6, respectively). Confocal micrographs illustrate RGCs and non-RGCs coexisting in the neurogenic niche. Inserts show a schematic view of the RGC to non-RGC transition. (D)E2-2 and E47 overexpression constructs decreased Ki67 immunoreactivity within progenitors (non-RGC) at 2 dpe (100 ± 5.6 vs. 61 ± 3.5 or 64.7 ± 4.1, respectively). shRNA constructs against E2-2 and E2A-induced opposite effects (100 ± 8 vs. 216 ± 13.2 or 161.9 ± 12.7, respectively). Confocal micrographs illustrate representative non-RGCs positive or negative for Ki67, respectively. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: C, 20 μm; D, 10 μm.
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Related In: Results  -  Collection

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Figure 4: E2-2 and E2A sequentially affect RGC differentiation and progenitor proliferation. (A) Representative illustration of a green fluorescence protein (GFP) plasmid electroporation into the lateral wall of the LV. (B)In vitro characterization of the shRNAs used in this study. shRNA against E2-2 or E2A decreased E2-2 or E2A expression (100 ± 18.9 vs. 17.2 ± 2.4 or 100 ± 20.2 vs. 20.9 ± 2.5, respectively) within neurosphere cultures, when co-nucleofected with E2-2 or E47 overexpression plasmids, respectively. (C)E2-2 and E47 overexpression constructs increased transition from RGCs (bipolar processes, apical connection to LV lumen) to non-RGCs (no processes, spherical morphologies, distant from the LV wall) at 2 dpe (100 ± 3.9 vs. 130.5 ± 3.0 or 139.7 ± 1.4, respectively). When shRNA plasmids against E2-2 or E2A were applied, most electroporated cells maintained a RGC morphology, as reflected by the reduced ratio of non-RGC cells compared to control conditions (100 ± 3 vs. 81 ± 6.4 or 82.9 ± 3.6, respectively). Confocal micrographs illustrate RGCs and non-RGCs coexisting in the neurogenic niche. Inserts show a schematic view of the RGC to non-RGC transition. (D)E2-2 and E47 overexpression constructs decreased Ki67 immunoreactivity within progenitors (non-RGC) at 2 dpe (100 ± 5.6 vs. 61 ± 3.5 or 64.7 ± 4.1, respectively). shRNA constructs against E2-2 and E2A-induced opposite effects (100 ± 8 vs. 216 ± 13.2 or 161.9 ± 12.7, respectively). Confocal micrographs illustrate representative non-RGCs positive or negative for Ki67, respectively. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions. Scale bars: C, 20 μm; D, 10 μm.
Mentions: We next performed further E-protein gain-of-function (GoF) and loss-of-function (LoF) experiments in the postnatal lateral SVZ, by means of plasmid electroporation (as above) (Figure 4A). We focused this functional characterization onto E2-2, which appears to be the most prominent E-protein expressed in the postnatal and adult SVZ (see above), and E2A, which is the best studied E-protein in CNS development [23,25,29,35-37].

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