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Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Masserdotti G, Gillotin S, Sutor B, Drechsel D, Irmler M, Jørgensen HF, Sass S, Theis FJ, Beckers J, Berninger B, Guillemot F, Götz M - Cell Stem Cell (2015)

Bottom Line: We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes.Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4.Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Physiological Genomics, Biomedical Center, University of Munich, 80336 Munich, Germany; Institute for Stem Cell Research, Helmholtz Centre Munich, 85764 Neuherberg, Germany.

No MeSH data available.


Temporal Analysis of Genome-wide Transcription Changes in Astrocyte Reprogramming(A) Schematic representation of the experimental procedure inducing the activation of Neurog2ERT2-IRES-DsRed or Ascl1ERT2-IRES-DsRed by tamoxifen (OHT indicated by uppermost black bars) for reprogramming astrocytes into neurons.(B, C, E, and F) Micrographs of astrocytes infected with the constructs indicated in red on the left side and immunostained for the astrocytic marker GFAP (green) and the neuronal marker βIII-tubulin (white). Scale bars, 100 μm.(D and G) Quantification of non-reprogrammed cells (GFAP) or reprogrammed cells (βIII-tubulin) without or with OHT 8 days post-induction (DPI). Mean ± SEM; n = 4 independent experiments; statistical test: two-tailed Mann-Whitney test (∗p < 0.05).(H) Schematic representation of the experimental procedure for genome-wide mRNA analysis.(I) Heatmap of genes regulated by both Neurog2ERT2 and Ascl1ERT2 within 24 hr after induction by OHT.(J) Venn diagram of genes regulated by Neurog2ERT2 or Ascl1ERT2 24 hr after OHT.(K and L) Real-time qPCR) analysis on selected candidates upon Neurog2ERT2 (K) or Ascl1ERT2 (L) induction by OHT for 24 hr. Mean ± SEM; n = 3 independent experiments.See also Figure S1, Table S1, Table S2, Table S3, Table S4, and Table S5.
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fig1: Temporal Analysis of Genome-wide Transcription Changes in Astrocyte Reprogramming(A) Schematic representation of the experimental procedure inducing the activation of Neurog2ERT2-IRES-DsRed or Ascl1ERT2-IRES-DsRed by tamoxifen (OHT indicated by uppermost black bars) for reprogramming astrocytes into neurons.(B, C, E, and F) Micrographs of astrocytes infected with the constructs indicated in red on the left side and immunostained for the astrocytic marker GFAP (green) and the neuronal marker βIII-tubulin (white). Scale bars, 100 μm.(D and G) Quantification of non-reprogrammed cells (GFAP) or reprogrammed cells (βIII-tubulin) without or with OHT 8 days post-induction (DPI). Mean ± SEM; n = 4 independent experiments; statistical test: two-tailed Mann-Whitney test (∗p < 0.05).(H) Schematic representation of the experimental procedure for genome-wide mRNA analysis.(I) Heatmap of genes regulated by both Neurog2ERT2 and Ascl1ERT2 within 24 hr after induction by OHT.(J) Venn diagram of genes regulated by Neurog2ERT2 or Ascl1ERT2 24 hr after OHT.(K and L) Real-time qPCR) analysis on selected candidates upon Neurog2ERT2 (K) or Ascl1ERT2 (L) induction by OHT for 24 hr. Mean ± SEM; n = 3 independent experiments.See also Figure S1, Table S1, Table S2, Table S3, Table S4, and Table S5.

Mentions: In order to investigate the early events of direct reprogramming, the cDNA of Neurog2 and Ascl1 was fused to the modified estrogen receptor ligand binding domain ERT2 (Raposo et al., 2015) and sub-cloned into a retroviral construct, together with the red fluorescent protein (DsRed-Expressed2, hereafter indicated as DsRed) (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002). Proliferating astrocytes were obtained from postnatal day (P)6–7 mouse cerebral cortex Gray Matter (GM), avoiding the White Matter (WM) and ventricular regions comprising endogenous neural stem cells (Imura et al., 2006). The purity of these cultures was previously assessed with various astrocytic markers and genetic fate mapping (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002) (see also Figures S1I and S1J). Moreover, cells infected with control retroviral vectors expressing GFP or DsRed showed a low proportion of Lewis X+ progenitors (3.9% ± 1.6% at day 2, Figures S1A–S1H) and did not generate any βIII-tubulin+ neurons (0%, 250 cells counted/experiment, n = 8). Likewise, Neurog2ERT2-transduced or Ascl1ERT2-transduced cells remained GFAP+ and generated virtually no neurons after 1 week without 4-hydroxy-tamoxifen (OHT) addition (Figures 1B and 1E; quantification in Figures 1D and 1G, 0% with Neurog2ERT2 and 0.8% with Ascl1ERT2; Figures 1D and 1G). Thus, these cultures contain largely non-neurogenic proliferating astrocytes.


Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Masserdotti G, Gillotin S, Sutor B, Drechsel D, Irmler M, Jørgensen HF, Sass S, Theis FJ, Beckers J, Berninger B, Guillemot F, Götz M - Cell Stem Cell (2015)

Temporal Analysis of Genome-wide Transcription Changes in Astrocyte Reprogramming(A) Schematic representation of the experimental procedure inducing the activation of Neurog2ERT2-IRES-DsRed or Ascl1ERT2-IRES-DsRed by tamoxifen (OHT indicated by uppermost black bars) for reprogramming astrocytes into neurons.(B, C, E, and F) Micrographs of astrocytes infected with the constructs indicated in red on the left side and immunostained for the astrocytic marker GFAP (green) and the neuronal marker βIII-tubulin (white). Scale bars, 100 μm.(D and G) Quantification of non-reprogrammed cells (GFAP) or reprogrammed cells (βIII-tubulin) without or with OHT 8 days post-induction (DPI). Mean ± SEM; n = 4 independent experiments; statistical test: two-tailed Mann-Whitney test (∗p < 0.05).(H) Schematic representation of the experimental procedure for genome-wide mRNA analysis.(I) Heatmap of genes regulated by both Neurog2ERT2 and Ascl1ERT2 within 24 hr after induction by OHT.(J) Venn diagram of genes regulated by Neurog2ERT2 or Ascl1ERT2 24 hr after OHT.(K and L) Real-time qPCR) analysis on selected candidates upon Neurog2ERT2 (K) or Ascl1ERT2 (L) induction by OHT for 24 hr. Mean ± SEM; n = 3 independent experiments.See also Figure S1, Table S1, Table S2, Table S3, Table S4, and Table S5.
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fig1: Temporal Analysis of Genome-wide Transcription Changes in Astrocyte Reprogramming(A) Schematic representation of the experimental procedure inducing the activation of Neurog2ERT2-IRES-DsRed or Ascl1ERT2-IRES-DsRed by tamoxifen (OHT indicated by uppermost black bars) for reprogramming astrocytes into neurons.(B, C, E, and F) Micrographs of astrocytes infected with the constructs indicated in red on the left side and immunostained for the astrocytic marker GFAP (green) and the neuronal marker βIII-tubulin (white). Scale bars, 100 μm.(D and G) Quantification of non-reprogrammed cells (GFAP) or reprogrammed cells (βIII-tubulin) without or with OHT 8 days post-induction (DPI). Mean ± SEM; n = 4 independent experiments; statistical test: two-tailed Mann-Whitney test (∗p < 0.05).(H) Schematic representation of the experimental procedure for genome-wide mRNA analysis.(I) Heatmap of genes regulated by both Neurog2ERT2 and Ascl1ERT2 within 24 hr after induction by OHT.(J) Venn diagram of genes regulated by Neurog2ERT2 or Ascl1ERT2 24 hr after OHT.(K and L) Real-time qPCR) analysis on selected candidates upon Neurog2ERT2 (K) or Ascl1ERT2 (L) induction by OHT for 24 hr. Mean ± SEM; n = 3 independent experiments.See also Figure S1, Table S1, Table S2, Table S3, Table S4, and Table S5.
Mentions: In order to investigate the early events of direct reprogramming, the cDNA of Neurog2 and Ascl1 was fused to the modified estrogen receptor ligand binding domain ERT2 (Raposo et al., 2015) and sub-cloned into a retroviral construct, together with the red fluorescent protein (DsRed-Expressed2, hereafter indicated as DsRed) (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002). Proliferating astrocytes were obtained from postnatal day (P)6–7 mouse cerebral cortex Gray Matter (GM), avoiding the White Matter (WM) and ventricular regions comprising endogenous neural stem cells (Imura et al., 2006). The purity of these cultures was previously assessed with various astrocytic markers and genetic fate mapping (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002) (see also Figures S1I and S1J). Moreover, cells infected with control retroviral vectors expressing GFP or DsRed showed a low proportion of Lewis X+ progenitors (3.9% ± 1.6% at day 2, Figures S1A–S1H) and did not generate any βIII-tubulin+ neurons (0%, 250 cells counted/experiment, n = 8). Likewise, Neurog2ERT2-transduced or Ascl1ERT2-transduced cells remained GFAP+ and generated virtually no neurons after 1 week without 4-hydroxy-tamoxifen (OHT) addition (Figures 1B and 1E; quantification in Figures 1D and 1G, 0% with Neurog2ERT2 and 0.8% with Ascl1ERT2; Figures 1D and 1G). Thus, these cultures contain largely non-neurogenic proliferating astrocytes.

Bottom Line: We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes.Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4.Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Physiological Genomics, Biomedical Center, University of Munich, 80336 Munich, Germany; Institute for Stem Cell Research, Helmholtz Centre Munich, 85764 Neuherberg, Germany.

No MeSH data available.