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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.


Quantification of PCNA immunofluorescence in Tbr2− and Tbr2+ NPCs in early versus late S-phase.(a, b) Representative examples of PCNA immunofluorescence (white, middle panels, z-stack of five 0.95-μm optical sections from the centre of the nucleus) of Tbr2− (left panels) and Tbr2+ (right panels) NPCs. Nuclei showing relatively homogeneous PCNA immunoreactivity with a predominantly small punctate pattern were considered as early S-phase (a), nuclei showing a more heterogeneous PCNA immunoreactivity with a few strong clusters were considered as late S-phase (b). Intensity of PCNA immunofluorescence is shown in pseudocolour in the lower panels (blue, low values (0=lowest); red, high values (255=highest). Scale bars, 10 μm. (c–f) Quantification of PCNA immunofluorescence of Tbr2− and Tbr2+ NPCs in early and late S-phase as defined in (a) and (b), respectively. Data are from the same cryosection per embryo and are from three embryos, each from a different litter. Numbers of nuclei analysed for embryos 1, 2, 3, were Tbr2− early S (13, 14, 10), Tbr2+ early S (13, 12, 7), Tbr2− late S (8, 9, 5), Tbr2+ late S (8, 9, 4). (c, d) Average PCNA immunofluorescence (AU, arbitrary units) of individual Tbr2− (circles) and Tbr2+ (triangles) nuclei in early (c) and late (d) S-phase, plotted in order of increasing values, with the highest Tbr2− and Tbr2+ value arbitrarily set to 1. Red, green, blue symbols indicate embryos 1, 2, 3, respectively. Data distribution of Tbr2− versus Tbr2+ nuclei in late S-phase (d), Kolmogorov–Smirnov P-value=0.002. (e) Average PCNA immunofluorescence per nucleus of Tbr2− (black) and Tbr2+ (magenta) nuclei in early (left) and late (right) S-phase. Data are the mean of the values from the three embryos shown in (c) and (d); error bars, s.d.; *P=0.028. (f) s.d. of PCNA immunofluorescence between the pixels of a given nucleus. For each embryo, the mean s.d. of Tbr2− nuclei (black) in early (left) and late (right) S-phase was arbitrarily set to 1, and the mean s.d. of Tbr2+ nuclei (magenta) was expressed relative to this. Data are the mean of the three embryos; error bars, s.d.; ***P=0.001.
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f6: Quantification of PCNA immunofluorescence in Tbr2− and Tbr2+ NPCs in early versus late S-phase.(a, b) Representative examples of PCNA immunofluorescence (white, middle panels, z-stack of five 0.95-μm optical sections from the centre of the nucleus) of Tbr2− (left panels) and Tbr2+ (right panels) NPCs. Nuclei showing relatively homogeneous PCNA immunoreactivity with a predominantly small punctate pattern were considered as early S-phase (a), nuclei showing a more heterogeneous PCNA immunoreactivity with a few strong clusters were considered as late S-phase (b). Intensity of PCNA immunofluorescence is shown in pseudocolour in the lower panels (blue, low values (0=lowest); red, high values (255=highest). Scale bars, 10 μm. (c–f) Quantification of PCNA immunofluorescence of Tbr2− and Tbr2+ NPCs in early and late S-phase as defined in (a) and (b), respectively. Data are from the same cryosection per embryo and are from three embryos, each from a different litter. Numbers of nuclei analysed for embryos 1, 2, 3, were Tbr2− early S (13, 14, 10), Tbr2+ early S (13, 12, 7), Tbr2− late S (8, 9, 5), Tbr2+ late S (8, 9, 4). (c, d) Average PCNA immunofluorescence (AU, arbitrary units) of individual Tbr2− (circles) and Tbr2+ (triangles) nuclei in early (c) and late (d) S-phase, plotted in order of increasing values, with the highest Tbr2− and Tbr2+ value arbitrarily set to 1. Red, green, blue symbols indicate embryos 1, 2, 3, respectively. Data distribution of Tbr2− versus Tbr2+ nuclei in late S-phase (d), Kolmogorov–Smirnov P-value=0.002. (e) Average PCNA immunofluorescence per nucleus of Tbr2− (black) and Tbr2+ (magenta) nuclei in early (left) and late (right) S-phase. Data are the mean of the values from the three embryos shown in (c) and (d); error bars, s.d.; *P=0.028. (f) s.d. of PCNA immunofluorescence between the pixels of a given nucleus. For each embryo, the mean s.d. of Tbr2− nuclei (black) in early (left) and late (right) S-phase was arbitrarily set to 1, and the mean s.d. of Tbr2+ nuclei (magenta) was expressed relative to this. Data are the mean of the three embryos; error bars, s.d.; ***P=0.001.

Mentions: In accordance with previous observations with non-neural cells4041, we considered the following: AP and BP nuclei exhibiting diffuse PCNA immunoreactivity to be in G1 or G2 (see Fig. 3c,d); nuclei exhibiting numerous small PCNA puncta above the level of diffuse PCNA immunoreactivity to be in early S-phase (Fig. 6a); and nuclei exhibiting strong, occasionally clustered PCNA puncta well above the level of diffuse PCNA immunoreactivity to be in late S-phase (Fig. 6b). Quantification of the average PCNA immunofluorescence of a nucleus showed that this value extended over a certain range for both early (Fig. 6c) and late (Fig. 6d) S-phase nuclei. Plotting these values in ascending order for late S-phase nuclei revealed a significant difference between Tbr2− and Tbr2+ nuclei (Fig. 6d), with the mean value of Tbr2+ nuclei being 18% higher than that of Tbr2− nuclei (Fig. 6e), whereas no significant difference was observed between early S-phase Tbr2− and Tbr2+ nuclei (Fig. 6c,e). Thus, nuclei with, on average, a shorter S-phase (BPs) appear to maintain PCNA immunoreactivity at late S-phase at higher levels than nuclei with, on average, a longer S-phase (APs), although the magnitude of this increase (1.2-fold change) was less than that of the reduction in S-phase duration (1.6-fold change) in BPs relative to APs (Table 1, see Supplementary Fig. S2 for proportions of NPC sub-populations). Moreover, the s.d. of PCNA immunofluorescence between the pixels of a given nucleus was similar for Tbr2− and Tbr2+ nuclei in early S-phase, but was significantly higher for Tbr2+ than for Tbr2− nuclei in late S-phase (Fig. 6f). This suggested that the variability of clustered PCNA immunoreactivity at late S-phase was greater in nuclei with, on average, a shorter S-phase (BPs) than nuclei with, on average, a longer S-phase (APs).


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)

Quantification of PCNA immunofluorescence in Tbr2− and Tbr2+ NPCs in early versus late S-phase.(a, b) Representative examples of PCNA immunofluorescence (white, middle panels, z-stack of five 0.95-μm optical sections from the centre of the nucleus) of Tbr2− (left panels) and Tbr2+ (right panels) NPCs. Nuclei showing relatively homogeneous PCNA immunoreactivity with a predominantly small punctate pattern were considered as early S-phase (a), nuclei showing a more heterogeneous PCNA immunoreactivity with a few strong clusters were considered as late S-phase (b). Intensity of PCNA immunofluorescence is shown in pseudocolour in the lower panels (blue, low values (0=lowest); red, high values (255=highest). Scale bars, 10 μm. (c–f) Quantification of PCNA immunofluorescence of Tbr2− and Tbr2+ NPCs in early and late S-phase as defined in (a) and (b), respectively. Data are from the same cryosection per embryo and are from three embryos, each from a different litter. Numbers of nuclei analysed for embryos 1, 2, 3, were Tbr2− early S (13, 14, 10), Tbr2+ early S (13, 12, 7), Tbr2− late S (8, 9, 5), Tbr2+ late S (8, 9, 4). (c, d) Average PCNA immunofluorescence (AU, arbitrary units) of individual Tbr2− (circles) and Tbr2+ (triangles) nuclei in early (c) and late (d) S-phase, plotted in order of increasing values, with the highest Tbr2− and Tbr2+ value arbitrarily set to 1. Red, green, blue symbols indicate embryos 1, 2, 3, respectively. Data distribution of Tbr2− versus Tbr2+ nuclei in late S-phase (d), Kolmogorov–Smirnov P-value=0.002. (e) Average PCNA immunofluorescence per nucleus of Tbr2− (black) and Tbr2+ (magenta) nuclei in early (left) and late (right) S-phase. Data are the mean of the values from the three embryos shown in (c) and (d); error bars, s.d.; *P=0.028. (f) s.d. of PCNA immunofluorescence between the pixels of a given nucleus. For each embryo, the mean s.d. of Tbr2− nuclei (black) in early (left) and late (right) S-phase was arbitrarily set to 1, and the mean s.d. of Tbr2+ nuclei (magenta) was expressed relative to this. Data are the mean of the three embryos; error bars, s.d.; ***P=0.001.
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f6: Quantification of PCNA immunofluorescence in Tbr2− and Tbr2+ NPCs in early versus late S-phase.(a, b) Representative examples of PCNA immunofluorescence (white, middle panels, z-stack of five 0.95-μm optical sections from the centre of the nucleus) of Tbr2− (left panels) and Tbr2+ (right panels) NPCs. Nuclei showing relatively homogeneous PCNA immunoreactivity with a predominantly small punctate pattern were considered as early S-phase (a), nuclei showing a more heterogeneous PCNA immunoreactivity with a few strong clusters were considered as late S-phase (b). Intensity of PCNA immunofluorescence is shown in pseudocolour in the lower panels (blue, low values (0=lowest); red, high values (255=highest). Scale bars, 10 μm. (c–f) Quantification of PCNA immunofluorescence of Tbr2− and Tbr2+ NPCs in early and late S-phase as defined in (a) and (b), respectively. Data are from the same cryosection per embryo and are from three embryos, each from a different litter. Numbers of nuclei analysed for embryos 1, 2, 3, were Tbr2− early S (13, 14, 10), Tbr2+ early S (13, 12, 7), Tbr2− late S (8, 9, 5), Tbr2+ late S (8, 9, 4). (c, d) Average PCNA immunofluorescence (AU, arbitrary units) of individual Tbr2− (circles) and Tbr2+ (triangles) nuclei in early (c) and late (d) S-phase, plotted in order of increasing values, with the highest Tbr2− and Tbr2+ value arbitrarily set to 1. Red, green, blue symbols indicate embryos 1, 2, 3, respectively. Data distribution of Tbr2− versus Tbr2+ nuclei in late S-phase (d), Kolmogorov–Smirnov P-value=0.002. (e) Average PCNA immunofluorescence per nucleus of Tbr2− (black) and Tbr2+ (magenta) nuclei in early (left) and late (right) S-phase. Data are the mean of the values from the three embryos shown in (c) and (d); error bars, s.d.; *P=0.028. (f) s.d. of PCNA immunofluorescence between the pixels of a given nucleus. For each embryo, the mean s.d. of Tbr2− nuclei (black) in early (left) and late (right) S-phase was arbitrarily set to 1, and the mean s.d. of Tbr2+ nuclei (magenta) was expressed relative to this. Data are the mean of the three embryos; error bars, s.d.; ***P=0.001.
Mentions: In accordance with previous observations with non-neural cells4041, we considered the following: AP and BP nuclei exhibiting diffuse PCNA immunoreactivity to be in G1 or G2 (see Fig. 3c,d); nuclei exhibiting numerous small PCNA puncta above the level of diffuse PCNA immunoreactivity to be in early S-phase (Fig. 6a); and nuclei exhibiting strong, occasionally clustered PCNA puncta well above the level of diffuse PCNA immunoreactivity to be in late S-phase (Fig. 6b). Quantification of the average PCNA immunofluorescence of a nucleus showed that this value extended over a certain range for both early (Fig. 6c) and late (Fig. 6d) S-phase nuclei. Plotting these values in ascending order for late S-phase nuclei revealed a significant difference between Tbr2− and Tbr2+ nuclei (Fig. 6d), with the mean value of Tbr2+ nuclei being 18% higher than that of Tbr2− nuclei (Fig. 6e), whereas no significant difference was observed between early S-phase Tbr2− and Tbr2+ nuclei (Fig. 6c,e). Thus, nuclei with, on average, a shorter S-phase (BPs) appear to maintain PCNA immunoreactivity at late S-phase at higher levels than nuclei with, on average, a longer S-phase (APs), although the magnitude of this increase (1.2-fold change) was less than that of the reduction in S-phase duration (1.6-fold change) in BPs relative to APs (Table 1, see Supplementary Fig. S2 for proportions of NPC sub-populations). Moreover, the s.d. of PCNA immunofluorescence between the pixels of a given nucleus was similar for Tbr2− and Tbr2+ nuclei in early S-phase, but was significantly higher for Tbr2+ than for Tbr2− nuclei in late S-phase (Fig. 6f). This suggested that the variability of clustered PCNA immunoreactivity at late S-phase was greater in nuclei with, on average, a shorter S-phase (BPs) than nuclei with, on average, a longer S-phase (APs).

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.