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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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Antigenic properties of radial glial as well as non-radial glial cells and confirmation of differentiation blockade by dnE47 overexpression. (A, B), At 2 days post electroporation (dpe), 40% of the labeled cells showed morphological criteria of radial glial cells (RGCs, i.e., bipolar processes, apical connection to lateral ventricle (LV) lumen), while 60% presenting a spherical morphology were distant from the LV and were defined as non-RGCs. Morphologically-defined RGCs express the NSC marker Vimentin (88.4 ± 0.7) and were EGFP positive in Hes5-EGFP reporter mice (94.2 ± 2.4), but negative in Ascl1-EGFP reporter mice, in which type-B or type-C cells are labelled, respectively [33,34]. In contrast, non-RGCs consisted of a heterogeneous population of type-C (i.e., Ascl1+, 53.6 ± 4.3) and type-A cells (i.e., Dcx+, 45.9 ± 0.2). Non-RGCs were frequently proliferative (i.e., Ki67+, 58.2 ± 5.7). (C) A subsequent characterization of cycling non-RGCs revealed that most Ki67+ cells were type-C cells (Ascl1+, 60.1 ± 4.4), whereas fewer were type-A cells (Dcx+, 22.9 ± 4.0). Notably, a minor population of cycling cells were double-positive for Ascl1 and Dcx (10.1 ± 1.4), probably representing a transition phase in between type-C and type-A phenotypes. (D–G) Antigenic assessment of RGC differentiation blockade following dnE47 overexpression. Electroporation of dnE47 in the postnatal SVZ reduced RGC differentiation, as revealed by the increased number of Vimentin+/RFP+ RGCs (D) and concomitant decrease of Ascl1+/RFP+(E) and Dcx+/RFP+(F) non-RGCs, respectively, when compared to an empty RFP control plasmid 2 dpe (Vimentin: 100 ± 3.2 vs. 150.5 ± 1.6; Ascl1: 100 ± 8.4 vs. 48.2 ± 3.1; Dcx: 100 ± 3.3 vs. 39.5 ± 6.3). This blockade of differentiation was further supported by an increase in Dcx+/RFP+ type-A cells retaining expression of the type-C cell marker Ascl1 (Dcx+Ascl1+, 100 ± 25.7 vs. 308.5 ± 50.3) ( G ). P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (D–G). Scale bars: A, 10 μm.
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Figure 2: Antigenic properties of radial glial as well as non-radial glial cells and confirmation of differentiation blockade by dnE47 overexpression. (A, B), At 2 days post electroporation (dpe), 40% of the labeled cells showed morphological criteria of radial glial cells (RGCs, i.e., bipolar processes, apical connection to lateral ventricle (LV) lumen), while 60% presenting a spherical morphology were distant from the LV and were defined as non-RGCs. Morphologically-defined RGCs express the NSC marker Vimentin (88.4 ± 0.7) and were EGFP positive in Hes5-EGFP reporter mice (94.2 ± 2.4), but negative in Ascl1-EGFP reporter mice, in which type-B or type-C cells are labelled, respectively [33,34]. In contrast, non-RGCs consisted of a heterogeneous population of type-C (i.e., Ascl1+, 53.6 ± 4.3) and type-A cells (i.e., Dcx+, 45.9 ± 0.2). Non-RGCs were frequently proliferative (i.e., Ki67+, 58.2 ± 5.7). (C) A subsequent characterization of cycling non-RGCs revealed that most Ki67+ cells were type-C cells (Ascl1+, 60.1 ± 4.4), whereas fewer were type-A cells (Dcx+, 22.9 ± 4.0). Notably, a minor population of cycling cells were double-positive for Ascl1 and Dcx (10.1 ± 1.4), probably representing a transition phase in between type-C and type-A phenotypes. (D–G) Antigenic assessment of RGC differentiation blockade following dnE47 overexpression. Electroporation of dnE47 in the postnatal SVZ reduced RGC differentiation, as revealed by the increased number of Vimentin+/RFP+ RGCs (D) and concomitant decrease of Ascl1+/RFP+(E) and Dcx+/RFP+(F) non-RGCs, respectively, when compared to an empty RFP control plasmid 2 dpe (Vimentin: 100 ± 3.2 vs. 150.5 ± 1.6; Ascl1: 100 ± 8.4 vs. 48.2 ± 3.1; Dcx: 100 ± 3.3 vs. 39.5 ± 6.3). This blockade of differentiation was further supported by an increase in Dcx+/RFP+ type-A cells retaining expression of the type-C cell marker Ascl1 (Dcx+Ascl1+, 100 ± 25.7 vs. 308.5 ± 50.3) ( G ). P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (D–G). Scale bars: A, 10 μm.

Mentions: To confirm the accuracy in monitoring RGC differentiation progression by electroporation and analysis of morphological criteria, we next performed an in depth antigenic characterization of RGCs and non-RGCs. At 2 days post-electroporation (2 dpe), RGCs were highly positive for type-B cell markers (i.e., Vimentin and Hes5-EGFP) and completely devoid of the type-C cell marker Ascl1 (Figure 2A,B). In contrast, non-RGCs were characterized as a mix of type-C (Ascl1+, 50%) and type-A (Dcx+, 50%) progenitors (Figure 2A,B). Approximately half of the non-RGCs were proliferating, as indicated by expression of Ki67 (Figure 2B). Those proliferating cells were mostly Ascl1+ type-C cells (~60%, Figure 2C, Additional file 1C), while only ~25% expressed the type-A cell marker Dcx (Figure 2C, Additional file 1D). Interestingly, ~10% of proliferating non-RGCs exhibited a transitory phenotype between type-C and type-A cell stages and were positive for both markers (Ascl1+Dcx+; Figure 2C).


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)

Antigenic properties of radial glial as well as non-radial glial cells and confirmation of differentiation blockade by dnE47 overexpression. (A, B), At 2 days post electroporation (dpe), 40% of the labeled cells showed morphological criteria of radial glial cells (RGCs, i.e., bipolar processes, apical connection to lateral ventricle (LV) lumen), while 60% presenting a spherical morphology were distant from the LV and were defined as non-RGCs. Morphologically-defined RGCs express the NSC marker Vimentin (88.4 ± 0.7) and were EGFP positive in Hes5-EGFP reporter mice (94.2 ± 2.4), but negative in Ascl1-EGFP reporter mice, in which type-B or type-C cells are labelled, respectively [33,34]. In contrast, non-RGCs consisted of a heterogeneous population of type-C (i.e., Ascl1+, 53.6 ± 4.3) and type-A cells (i.e., Dcx+, 45.9 ± 0.2). Non-RGCs were frequently proliferative (i.e., Ki67+, 58.2 ± 5.7). (C) A subsequent characterization of cycling non-RGCs revealed that most Ki67+ cells were type-C cells (Ascl1+, 60.1 ± 4.4), whereas fewer were type-A cells (Dcx+, 22.9 ± 4.0). Notably, a minor population of cycling cells were double-positive for Ascl1 and Dcx (10.1 ± 1.4), probably representing a transition phase in between type-C and type-A phenotypes. (D–G) Antigenic assessment of RGC differentiation blockade following dnE47 overexpression. Electroporation of dnE47 in the postnatal SVZ reduced RGC differentiation, as revealed by the increased number of Vimentin+/RFP+ RGCs (D) and concomitant decrease of Ascl1+/RFP+(E) and Dcx+/RFP+(F) non-RGCs, respectively, when compared to an empty RFP control plasmid 2 dpe (Vimentin: 100 ± 3.2 vs. 150.5 ± 1.6; Ascl1: 100 ± 8.4 vs. 48.2 ± 3.1; Dcx: 100 ± 3.3 vs. 39.5 ± 6.3). This blockade of differentiation was further supported by an increase in Dcx+/RFP+ type-A cells retaining expression of the type-C cell marker Ascl1 (Dcx+Ascl1+, 100 ± 25.7 vs. 308.5 ± 50.3) ( G ). P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (D–G). Scale bars: A, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Antigenic properties of radial glial as well as non-radial glial cells and confirmation of differentiation blockade by dnE47 overexpression. (A, B), At 2 days post electroporation (dpe), 40% of the labeled cells showed morphological criteria of radial glial cells (RGCs, i.e., bipolar processes, apical connection to lateral ventricle (LV) lumen), while 60% presenting a spherical morphology were distant from the LV and were defined as non-RGCs. Morphologically-defined RGCs express the NSC marker Vimentin (88.4 ± 0.7) and were EGFP positive in Hes5-EGFP reporter mice (94.2 ± 2.4), but negative in Ascl1-EGFP reporter mice, in which type-B or type-C cells are labelled, respectively [33,34]. In contrast, non-RGCs consisted of a heterogeneous population of type-C (i.e., Ascl1+, 53.6 ± 4.3) and type-A cells (i.e., Dcx+, 45.9 ± 0.2). Non-RGCs were frequently proliferative (i.e., Ki67+, 58.2 ± 5.7). (C) A subsequent characterization of cycling non-RGCs revealed that most Ki67+ cells were type-C cells (Ascl1+, 60.1 ± 4.4), whereas fewer were type-A cells (Dcx+, 22.9 ± 4.0). Notably, a minor population of cycling cells were double-positive for Ascl1 and Dcx (10.1 ± 1.4), probably representing a transition phase in between type-C and type-A phenotypes. (D–G) Antigenic assessment of RGC differentiation blockade following dnE47 overexpression. Electroporation of dnE47 in the postnatal SVZ reduced RGC differentiation, as revealed by the increased number of Vimentin+/RFP+ RGCs (D) and concomitant decrease of Ascl1+/RFP+(E) and Dcx+/RFP+(F) non-RGCs, respectively, when compared to an empty RFP control plasmid 2 dpe (Vimentin: 100 ± 3.2 vs. 150.5 ± 1.6; Ascl1: 100 ± 8.4 vs. 48.2 ± 3.1; Dcx: 100 ± 3.3 vs. 39.5 ± 6.3). This blockade of differentiation was further supported by an increase in Dcx+/RFP+ type-A cells retaining expression of the type-C cell marker Ascl1 (Dcx+Ascl1+, 100 ± 25.7 vs. 308.5 ± 50.3) ( G ). P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (D–G). Scale bars: A, 10 μm.
Mentions: To confirm the accuracy in monitoring RGC differentiation progression by electroporation and analysis of morphological criteria, we next performed an in depth antigenic characterization of RGCs and non-RGCs. At 2 days post-electroporation (2 dpe), RGCs were highly positive for type-B cell markers (i.e., Vimentin and Hes5-EGFP) and completely devoid of the type-C cell marker Ascl1 (Figure 2A,B). In contrast, non-RGCs were characterized as a mix of type-C (Ascl1+, 50%) and type-A (Dcx+, 50%) progenitors (Figure 2A,B). Approximately half of the non-RGCs were proliferating, as indicated by expression of Ki67 (Figure 2B). Those proliferating cells were mostly Ascl1+ type-C cells (~60%, Figure 2C, Additional file 1C), while only ~25% expressed the type-A cell marker Dcx (Figure 2C, Additional file 1D). Interestingly, ~10% of proliferating non-RGCs exhibited a transitory phenotype between type-C and type-A cell stages and were positive for both markers (Ascl1+Dcx+; Figure 2C).

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