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Neural stem and progenitor cells shorten S-phase on commitment to neuron production.

Arai Y, Pulvers JN, Haffner C, Schilling B, Nüsslein I, Calegari F, Huttner WB - Nat Commun (2011)

Bottom Line: We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors.Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling.Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
During mammalian cerebral cortex development, the G1-phase of the cell cycle is known to lengthen, but it has been unclear which neural stem and progenitor cells are affected. In this paper, we develop a novel approach to determine cell-cycle parameters in specific classes of neural stem and progenitor cells, identified by molecular markers rather than location. We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors. Unexpectedly, expanding apical and basal progenitors exhibit a substantially longer S-phase than apical and basal progenitors committed to neuron production. Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling. Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

No MeSH data available.


Identification of S-phase NPC nuclei by PCNA immunostaining.(a, b, c, d) Pax6 (magenta) or Tbr2 (magenta), PCNA (blue) and DAPI staining (c, d, white). Individual nuclei indicated by arrowheads in (a) and (b) are shown at higher magnification in (c) and (d), respectively; white arrowheads, S-phase nuclei showing punctate PCNA immunoreactivity; yellow arrowheads, G1- or G2-phase nuclei showing homogeneous PCNA immunoreactivity. All Pax6+ nuclei (a) and all Tbr2+ nuclei (b) that show punctate PCNA immunoreactivity are indicated by white dots in the respective right panel. Scale bars, 50 μm (a, b) and 10 μm (c, d). (e) Percentage of AP and BP nuclei that are in S-phase as revealed by PCNA immunostaining. Data show the percentage value of (BP) total Tbr2+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of neurons, and of (AP) total Pax6+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of BPs, and are the mean of four 225-μm-wide fields, each from a different brain and litter; error bars indicate s.d.; **P<0.01. (a–e) Images in (a–d) and data in (e) are from embryos subjected to cumulative EdU labelling for 5 and 3–9 h, respectively.
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f3: Identification of S-phase NPC nuclei by PCNA immunostaining.(a, b, c, d) Pax6 (magenta) or Tbr2 (magenta), PCNA (blue) and DAPI staining (c, d, white). Individual nuclei indicated by arrowheads in (a) and (b) are shown at higher magnification in (c) and (d), respectively; white arrowheads, S-phase nuclei showing punctate PCNA immunoreactivity; yellow arrowheads, G1- or G2-phase nuclei showing homogeneous PCNA immunoreactivity. All Pax6+ nuclei (a) and all Tbr2+ nuclei (b) that show punctate PCNA immunoreactivity are indicated by white dots in the respective right panel. Scale bars, 50 μm (a, b) and 10 μm (c, d). (e) Percentage of AP and BP nuclei that are in S-phase as revealed by PCNA immunostaining. Data show the percentage value of (BP) total Tbr2+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of neurons, and of (AP) total Pax6+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of BPs, and are the mean of four 225-μm-wide fields, each from a different brain and litter; error bars indicate s.d.; **P<0.01. (a–e) Images in (a–d) and data in (e) are from embryos subjected to cumulative EdU labelling for 5 and 3–9 h, respectively.

Mentions: We sought to corroborate the difference between APs and BPs with regard to the S-phase proportion relative to the total cell cycle by determining the proportion of NPCs in S-phase using an independent method, that is, proliferating cell nuclear antigen (PCNA) immunostaining. Cell nuclei in S-phase typically exhibit a punctate pattern of PCNA immunoreactivity, which reflects sites of DNA replication, whereas nuclei in G1 and G2 show diffuse PCNA immunoreactivity (Fig. 3a–d)3738. Determination of the percentage of Pax6+ (Fig. 3a) and Tbr2+ (Fig. 3b) nuclei in S-phase by PCNA immunostaining, together with correction for the contribution of neurons to the Tbr2+ nuclei and of BPs to the Pax6+ nuclei (Fig. 1b,g) as described in the Methods, allowed us to calculate the percentage of APs and BPs in S-phase (30±4 and 14±4%, respectively; Fig. 3e). Very similar data to that shown in Figure 3e (EdU-labelled samples) were obtained when embryos not exposed to EdU were analysed (APs 26±6% (n=3); BPs 12–14% (n=2)). These data compare well with the mean proportion of NPCs in S-phase as determined from the intercept of the cumulative EdU-labelling curves with the y axis (Fig. 2e).


Neural stem and progenitor cells shorten S-phase on commitment to neuron production.

Arai Y, Pulvers JN, Haffner C, Schilling B, Nüsslein I, Calegari F, Huttner WB - Nat Commun (2011)

Identification of S-phase NPC nuclei by PCNA immunostaining.(a, b, c, d) Pax6 (magenta) or Tbr2 (magenta), PCNA (blue) and DAPI staining (c, d, white). Individual nuclei indicated by arrowheads in (a) and (b) are shown at higher magnification in (c) and (d), respectively; white arrowheads, S-phase nuclei showing punctate PCNA immunoreactivity; yellow arrowheads, G1- or G2-phase nuclei showing homogeneous PCNA immunoreactivity. All Pax6+ nuclei (a) and all Tbr2+ nuclei (b) that show punctate PCNA immunoreactivity are indicated by white dots in the respective right panel. Scale bars, 50 μm (a, b) and 10 μm (c, d). (e) Percentage of AP and BP nuclei that are in S-phase as revealed by PCNA immunostaining. Data show the percentage value of (BP) total Tbr2+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of neurons, and of (AP) total Pax6+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of BPs, and are the mean of four 225-μm-wide fields, each from a different brain and litter; error bars indicate s.d.; **P<0.01. (a–e) Images in (a–d) and data in (e) are from embryos subjected to cumulative EdU labelling for 5 and 3–9 h, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Identification of S-phase NPC nuclei by PCNA immunostaining.(a, b, c, d) Pax6 (magenta) or Tbr2 (magenta), PCNA (blue) and DAPI staining (c, d, white). Individual nuclei indicated by arrowheads in (a) and (b) are shown at higher magnification in (c) and (d), respectively; white arrowheads, S-phase nuclei showing punctate PCNA immunoreactivity; yellow arrowheads, G1- or G2-phase nuclei showing homogeneous PCNA immunoreactivity. All Pax6+ nuclei (a) and all Tbr2+ nuclei (b) that show punctate PCNA immunoreactivity are indicated by white dots in the respective right panel. Scale bars, 50 μm (a, b) and 10 μm (c, d). (e) Percentage of AP and BP nuclei that are in S-phase as revealed by PCNA immunostaining. Data show the percentage value of (BP) total Tbr2+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of neurons, and of (AP) total Pax6+ nuclei in VZ+SVZ showing punctate PCNA immunoreactivity, corrected for the contribution of BPs, and are the mean of four 225-μm-wide fields, each from a different brain and litter; error bars indicate s.d.; **P<0.01. (a–e) Images in (a–d) and data in (e) are from embryos subjected to cumulative EdU labelling for 5 and 3–9 h, respectively.
Mentions: We sought to corroborate the difference between APs and BPs with regard to the S-phase proportion relative to the total cell cycle by determining the proportion of NPCs in S-phase using an independent method, that is, proliferating cell nuclear antigen (PCNA) immunostaining. Cell nuclei in S-phase typically exhibit a punctate pattern of PCNA immunoreactivity, which reflects sites of DNA replication, whereas nuclei in G1 and G2 show diffuse PCNA immunoreactivity (Fig. 3a–d)3738. Determination of the percentage of Pax6+ (Fig. 3a) and Tbr2+ (Fig. 3b) nuclei in S-phase by PCNA immunostaining, together with correction for the contribution of neurons to the Tbr2+ nuclei and of BPs to the Pax6+ nuclei (Fig. 1b,g) as described in the Methods, allowed us to calculate the percentage of APs and BPs in S-phase (30±4 and 14±4%, respectively; Fig. 3e). Very similar data to that shown in Figure 3e (EdU-labelled samples) were obtained when embryos not exposed to EdU were analysed (APs 26±6% (n=3); BPs 12–14% (n=2)). These data compare well with the mean proportion of NPCs in S-phase as determined from the intercept of the cumulative EdU-labelling curves with the y axis (Fig. 2e).

Bottom Line: We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors.Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling.Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
During mammalian cerebral cortex development, the G1-phase of the cell cycle is known to lengthen, but it has been unclear which neural stem and progenitor cells are affected. In this paper, we develop a novel approach to determine cell-cycle parameters in specific classes of neural stem and progenitor cells, identified by molecular markers rather than location. We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors. Unexpectedly, expanding apical and basal progenitors exhibit a substantially longer S-phase than apical and basal progenitors committed to neuron production. Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling. Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

No MeSH data available.