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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 DNA synthesis rate by EdU incorporation in Tis21-GFP− and Tis21-GFP+ NPCs.Following a 30-min EdU pulse labelling (two litters), cortical cells were dissociated, analysed by flow cytometry and sorted for Tis21-GFP fluorescence. Tis21-GFP− and Tis21-GFP+ cells were then separately analysed for DNA content and EdU incorporation in another round of flow cytometry, as shown in the panels. (a, b) Granularity (side scatter, SSC; AU, arbitrary units) versus EdU fluorescence of single Tis21-GFP− (a, 8,053 cells) and Tis21-GFP+ (b, 8,029 cells) cells. Dashed lines, overtly EdU-incorporating cells quantified in (c). Plots show one of the two litters analysed. (c) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey column) and Tis21-GFP+ (green column) cells. For each litter, the mean EdU fluorescence of Tis21-GFP− cells was arbitrarily set to 100, and the mean EdU fluorescence of Tis21-GFP+ cells was expressed relative to this. Data are the mean of two litters, dots indicate the two individual values. (d, e) Cell number versus DNA content of the single Tis21-GFP− (d) and Tis21-GFP+ (e) cells shown in (a) and (b), respectively. Boxes indicate the sub-populations of cells in G0/G1, early S, late S and G2+M phases, as defined by DNA content; cells in early and late S-phases were analysed for SSC versus EdU fluorescence in (g–j). (f) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey columns) and Tis21-GFP+ (green columns) cells in early (g, i) and late (h, j) S-phase, as defined by DNA content (d, e) and after analysis of SSC versus EdU fluorescence (g–j). Quantification as in (c). (g–j) Granularity (SSC) versus EdU fluorescence of single Tis21-GFP− (g, h) and Tis21-GFP+ (i, j) cells in early (g, i) and late (h, j) S phase shown in (d) and (e), respectively. Dashed lines, overtly EdU-incorporating cells quantified in (f). Plots show one of the two litters analysed. AU, arbitrary units.
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f5: Quantification of DNA synthesis rate by EdU incorporation in Tis21-GFP− and Tis21-GFP+ NPCs.Following a 30-min EdU pulse labelling (two litters), cortical cells were dissociated, analysed by flow cytometry and sorted for Tis21-GFP fluorescence. Tis21-GFP− and Tis21-GFP+ cells were then separately analysed for DNA content and EdU incorporation in another round of flow cytometry, as shown in the panels. (a, b) Granularity (side scatter, SSC; AU, arbitrary units) versus EdU fluorescence of single Tis21-GFP− (a, 8,053 cells) and Tis21-GFP+ (b, 8,029 cells) cells. Dashed lines, overtly EdU-incorporating cells quantified in (c). Plots show one of the two litters analysed. (c) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey column) and Tis21-GFP+ (green column) cells. For each litter, the mean EdU fluorescence of Tis21-GFP− cells was arbitrarily set to 100, and the mean EdU fluorescence of Tis21-GFP+ cells was expressed relative to this. Data are the mean of two litters, dots indicate the two individual values. (d, e) Cell number versus DNA content of the single Tis21-GFP− (d) and Tis21-GFP+ (e) cells shown in (a) and (b), respectively. Boxes indicate the sub-populations of cells in G0/G1, early S, late S and G2+M phases, as defined by DNA content; cells in early and late S-phases were analysed for SSC versus EdU fluorescence in (g–j). (f) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey columns) and Tis21-GFP+ (green columns) cells in early (g, i) and late (h, j) S-phase, as defined by DNA content (d, e) and after analysis of SSC versus EdU fluorescence (g–j). Quantification as in (c). (g–j) Granularity (SSC) versus EdU fluorescence of single Tis21-GFP− (g, h) and Tis21-GFP+ (i, j) cells in early (g, i) and late (h, j) S phase shown in (d) and (e), respectively. Dashed lines, overtly EdU-incorporating cells quantified in (f). Plots show one of the two litters analysed. AU, arbitrary units.

Mentions: To investigate possible mechanisms underlying the S-phase shortening in Tis21-GFP+ compared with Tis21-GFP− NPCs, we performed EdU pulse-labelling for 30 min in vivo and analysed dissociated single Tis21-GFP− and Tis21-GFP+ NPCs, separated by fluorescence-activated cell sorting (FACS), by flow cytometry. Comparison of the overtly EdU-incorporating Tis21-GFP− (Fig. 5a, dashed line) and Tis21-GFP+ (Fig. 5b, dashed line) NPC populations revealed a 20% increase in the rate of EdU incorporation in Tis21-GFP+ cells (Fig. 5c). This increase was corroborated when only the overtly EdU-incorporating NPCs in S-phase, as defined by DNA content (Fig. 5d,e), were quantified, and observed for cells in early and late S-phase (Fig. 5f; Fig. 5g,h for Tis21-GFP−; Fig. 5i,j for Tis21-GFP+). Thus, Tis21-GFP+ NPCs exhibited a higher rate of DNA synthesis than Tis21-GFP− NPCs, although the magnitude of this increase (1.2-fold change) was clearly less than that of the reduction in S-phase duration (3.3-fold change) in Tis21-GFP+ compared with Tis21-GFP− NPCs (Fig. 4f, Table 1, see Supplementary Fig. S2 for proportions of NPC sub-populations).


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 DNA synthesis rate by EdU incorporation in Tis21-GFP− and Tis21-GFP+ NPCs.Following a 30-min EdU pulse labelling (two litters), cortical cells were dissociated, analysed by flow cytometry and sorted for Tis21-GFP fluorescence. Tis21-GFP− and Tis21-GFP+ cells were then separately analysed for DNA content and EdU incorporation in another round of flow cytometry, as shown in the panels. (a, b) Granularity (side scatter, SSC; AU, arbitrary units) versus EdU fluorescence of single Tis21-GFP− (a, 8,053 cells) and Tis21-GFP+ (b, 8,029 cells) cells. Dashed lines, overtly EdU-incorporating cells quantified in (c). Plots show one of the two litters analysed. (c) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey column) and Tis21-GFP+ (green column) cells. For each litter, the mean EdU fluorescence of Tis21-GFP− cells was arbitrarily set to 100, and the mean EdU fluorescence of Tis21-GFP+ cells was expressed relative to this. Data are the mean of two litters, dots indicate the two individual values. (d, e) Cell number versus DNA content of the single Tis21-GFP− (d) and Tis21-GFP+ (e) cells shown in (a) and (b), respectively. Boxes indicate the sub-populations of cells in G0/G1, early S, late S and G2+M phases, as defined by DNA content; cells in early and late S-phases were analysed for SSC versus EdU fluorescence in (g–j). (f) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey columns) and Tis21-GFP+ (green columns) cells in early (g, i) and late (h, j) S-phase, as defined by DNA content (d, e) and after analysis of SSC versus EdU fluorescence (g–j). Quantification as in (c). (g–j) Granularity (SSC) versus EdU fluorescence of single Tis21-GFP− (g, h) and Tis21-GFP+ (i, j) cells in early (g, i) and late (h, j) S phase shown in (d) and (e), respectively. Dashed lines, overtly EdU-incorporating cells quantified in (f). Plots show one of the two litters analysed. AU, arbitrary units.
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Related In: Results  -  Collection

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f5: Quantification of DNA synthesis rate by EdU incorporation in Tis21-GFP− and Tis21-GFP+ NPCs.Following a 30-min EdU pulse labelling (two litters), cortical cells were dissociated, analysed by flow cytometry and sorted for Tis21-GFP fluorescence. Tis21-GFP− and Tis21-GFP+ cells were then separately analysed for DNA content and EdU incorporation in another round of flow cytometry, as shown in the panels. (a, b) Granularity (side scatter, SSC; AU, arbitrary units) versus EdU fluorescence of single Tis21-GFP− (a, 8,053 cells) and Tis21-GFP+ (b, 8,029 cells) cells. Dashed lines, overtly EdU-incorporating cells quantified in (c). Plots show one of the two litters analysed. (c) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey column) and Tis21-GFP+ (green column) cells. For each litter, the mean EdU fluorescence of Tis21-GFP− cells was arbitrarily set to 100, and the mean EdU fluorescence of Tis21-GFP+ cells was expressed relative to this. Data are the mean of two litters, dots indicate the two individual values. (d, e) Cell number versus DNA content of the single Tis21-GFP− (d) and Tis21-GFP+ (e) cells shown in (a) and (b), respectively. Boxes indicate the sub-populations of cells in G0/G1, early S, late S and G2+M phases, as defined by DNA content; cells in early and late S-phases were analysed for SSC versus EdU fluorescence in (g–j). (f) Mean EdU fluorescence of the overtly EdU-incorporating single Tis21-GFP− (grey columns) and Tis21-GFP+ (green columns) cells in early (g, i) and late (h, j) S-phase, as defined by DNA content (d, e) and after analysis of SSC versus EdU fluorescence (g–j). Quantification as in (c). (g–j) Granularity (SSC) versus EdU fluorescence of single Tis21-GFP− (g, h) and Tis21-GFP+ (i, j) cells in early (g, i) and late (h, j) S phase shown in (d) and (e), respectively. Dashed lines, overtly EdU-incorporating cells quantified in (f). Plots show one of the two litters analysed. AU, arbitrary units.
Mentions: To investigate possible mechanisms underlying the S-phase shortening in Tis21-GFP+ compared with Tis21-GFP− NPCs, we performed EdU pulse-labelling for 30 min in vivo and analysed dissociated single Tis21-GFP− and Tis21-GFP+ NPCs, separated by fluorescence-activated cell sorting (FACS), by flow cytometry. Comparison of the overtly EdU-incorporating Tis21-GFP− (Fig. 5a, dashed line) and Tis21-GFP+ (Fig. 5b, dashed line) NPC populations revealed a 20% increase in the rate of EdU incorporation in Tis21-GFP+ cells (Fig. 5c). This increase was corroborated when only the overtly EdU-incorporating NPCs in S-phase, as defined by DNA content (Fig. 5d,e), were quantified, and observed for cells in early and late S-phase (Fig. 5f; Fig. 5g,h for Tis21-GFP−; Fig. 5i,j for Tis21-GFP+). Thus, Tis21-GFP+ NPCs exhibited a higher rate of DNA synthesis than Tis21-GFP− NPCs, although the magnitude of this increase (1.2-fold change) was clearly less than that of the reduction in S-phase duration (3.3-fold change) in Tis21-GFP+ compared with Tis21-GFP− NPCs (Fig. 4f, Table 1, see Supplementary Fig. S2 for proportions of NPC sub-populations).

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.