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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.


Related in: MedlinePlus

Cumulative EdU labelling of proliferative and neurogenic APs and BPs.(a, b) Pax6 (magenta), Tbr2 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual AP nuclei indicated by arrows/arrowheads in (a) are shown at higher magnification in (b) (asterisks); white arrowheads, Pax6+/Tbr2−/Tis21-GFP−/EdU− nucleus; white arrows, Pax6+/Tbr2−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Pax6+/Tbr2−/Tis21-GFP+/EdU− nucleus; yellow arrows, Pax6+/Tbr2−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (a) and 10 μm (b). (c, d) Tbr2 (magenta), Tbr1 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual BP nuclei indicated by arrows/arrowheads in (c) are shown at higher magnification in (d) (asterisks); white arrowheads, Tbr2+/Tbr1−/Tis21-GFP−/EdU− nucleus; white arrows, Tbr2+/Tbr1−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Tbr2+/Tbr1−/Tis21-GFP+/EdU− nucleus; yellow arrows, Tbr2+/Tbr1−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (c) and 10 μm (d). (e, f) Proportion of EdU-labelled NPC nuclei (EdU labelling index) after cumulative EdU labelling for 0.5, 1, 2, 3, 5, 9, 12, 18, 24 and 29 h. The EdU labelling index was separately determined for the various AP (Pax6/Tbr2/GFP/EdU staining as in (a)) and BP (Tbr2/Tbr1/GFP/EdU staining as in (c)) populations, as follows: total APs (e, Tbr2−/Pax6+, blue circles), total BPs (e, Tbr1−/Tbr2+, red squares), proliferative APs (f, Tbr2−/Pax6+/Tis21-GFP−, blue diamonds), BP-genic/neurogenic APs (f, Tbr2−/Pax6+/Tis21-GFP+, purple squares), proliferative BPs (f, Tbr1−/Tbr2+/Tis21-GFP−, red triangles) and neurogenic BPs (f, Tbr1−/Tbr2+/Tis21-GFP+, green crosses). Data are the mean of three 225-μm-wide fields (except for the 1-, 9- and 29-h time points, which are from five, two and one field(s), respectively), each from a different brain and litter; error bars indicate s.e.m. or the range of the two individual values. Dashed lines and colour-coded arrowheads indicate the growth fraction; colour-coded arrows indicate the time point at which the labelling index reaches a plateau (TC–TS).
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f2: Cumulative EdU labelling of proliferative and neurogenic APs and BPs.(a, b) Pax6 (magenta), Tbr2 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual AP nuclei indicated by arrows/arrowheads in (a) are shown at higher magnification in (b) (asterisks); white arrowheads, Pax6+/Tbr2−/Tis21-GFP−/EdU− nucleus; white arrows, Pax6+/Tbr2−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Pax6+/Tbr2−/Tis21-GFP+/EdU− nucleus; yellow arrows, Pax6+/Tbr2−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (a) and 10 μm (b). (c, d) Tbr2 (magenta), Tbr1 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual BP nuclei indicated by arrows/arrowheads in (c) are shown at higher magnification in (d) (asterisks); white arrowheads, Tbr2+/Tbr1−/Tis21-GFP−/EdU− nucleus; white arrows, Tbr2+/Tbr1−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Tbr2+/Tbr1−/Tis21-GFP+/EdU− nucleus; yellow arrows, Tbr2+/Tbr1−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (c) and 10 μm (d). (e, f) Proportion of EdU-labelled NPC nuclei (EdU labelling index) after cumulative EdU labelling for 0.5, 1, 2, 3, 5, 9, 12, 18, 24 and 29 h. The EdU labelling index was separately determined for the various AP (Pax6/Tbr2/GFP/EdU staining as in (a)) and BP (Tbr2/Tbr1/GFP/EdU staining as in (c)) populations, as follows: total APs (e, Tbr2−/Pax6+, blue circles), total BPs (e, Tbr1−/Tbr2+, red squares), proliferative APs (f, Tbr2−/Pax6+/Tis21-GFP−, blue diamonds), BP-genic/neurogenic APs (f, Tbr2−/Pax6+/Tis21-GFP+, purple squares), proliferative BPs (f, Tbr1−/Tbr2+/Tis21-GFP−, red triangles) and neurogenic BPs (f, Tbr1−/Tbr2+/Tis21-GFP+, green crosses). Data are the mean of three 225-μm-wide fields (except for the 1-, 9- and 29-h time points, which are from five, two and one field(s), respectively), each from a different brain and litter; error bars indicate s.e.m. or the range of the two individual values. Dashed lines and colour-coded arrowheads indicate the growth fraction; colour-coded arrows indicate the time point at which the labelling index reaches a plateau (TC–TS).

Mentions: We next used the above information to determine the cell-cycle parameters of Tis21-GFP-negative (Tis21-GFP−) and -positive (Tis21-GFP+) APs and BPs, using cumulative labelling with 5-ethynyl-2-deoxyuridine (EdU). As described in Supplementary Note S2, it was appropriate to use EdU instead of 5-bromo-2-deoxyuridine (BrdU; Supplementary Figs S3 and S4). Cumulative EdU labelling was performed in conjunction with triple immunostaining for either Pax6, Tbr2 plus Tis21-GFP (Fig. 2a,b) or Tbr2, Tbr1 plus Tis21-GFP (Fig. 2c,d) to determine the cell-cycle parameters for APs (Tbr2−/Pax6+) and BPs (Tbr1−/Tbr2+), respectively, and to distinguish between proliferative (Tis21-GFP−) and neurogenic (Tis21-GFP+) NPC sub-populations. As shown in Figure 2e and summarized in Table 1, this revealed that the length of G2+M+G1 (TC–TS), as indicated by the time point at which the EdU labelling index reached the plateau, was longer for the total population of BPs (23.3 h) than APs (14.1 h). In contrast, the proportion of the cell cycle comprising S-phase, as indicated by the intercept of the cumulative EdU labelling curve with the y axis, was smaller for BPs (12%) than for APs (26%). The growth fraction was nearly 100% for both, APs and BPs. Calculation of the length of S-phase (TS) and the total cell cycle (TC)24 yielded values of 5.0 and 19.1 h for APs and 3.2 and 26.5 h for BPs. The present TC data determined by cumulative EdU labelling are very similar to those determined by live imaging in organotypic slice culture720.


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)

Cumulative EdU labelling of proliferative and neurogenic APs and BPs.(a, b) Pax6 (magenta), Tbr2 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual AP nuclei indicated by arrows/arrowheads in (a) are shown at higher magnification in (b) (asterisks); white arrowheads, Pax6+/Tbr2−/Tis21-GFP−/EdU− nucleus; white arrows, Pax6+/Tbr2−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Pax6+/Tbr2−/Tis21-GFP+/EdU− nucleus; yellow arrows, Pax6+/Tbr2−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (a) and 10 μm (b). (c, d) Tbr2 (magenta), Tbr1 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual BP nuclei indicated by arrows/arrowheads in (c) are shown at higher magnification in (d) (asterisks); white arrowheads, Tbr2+/Tbr1−/Tis21-GFP−/EdU− nucleus; white arrows, Tbr2+/Tbr1−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Tbr2+/Tbr1−/Tis21-GFP+/EdU− nucleus; yellow arrows, Tbr2+/Tbr1−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (c) and 10 μm (d). (e, f) Proportion of EdU-labelled NPC nuclei (EdU labelling index) after cumulative EdU labelling for 0.5, 1, 2, 3, 5, 9, 12, 18, 24 and 29 h. The EdU labelling index was separately determined for the various AP (Pax6/Tbr2/GFP/EdU staining as in (a)) and BP (Tbr2/Tbr1/GFP/EdU staining as in (c)) populations, as follows: total APs (e, Tbr2−/Pax6+, blue circles), total BPs (e, Tbr1−/Tbr2+, red squares), proliferative APs (f, Tbr2−/Pax6+/Tis21-GFP−, blue diamonds), BP-genic/neurogenic APs (f, Tbr2−/Pax6+/Tis21-GFP+, purple squares), proliferative BPs (f, Tbr1−/Tbr2+/Tis21-GFP−, red triangles) and neurogenic BPs (f, Tbr1−/Tbr2+/Tis21-GFP+, green crosses). Data are the mean of three 225-μm-wide fields (except for the 1-, 9- and 29-h time points, which are from five, two and one field(s), respectively), each from a different brain and litter; error bars indicate s.e.m. or the range of the two individual values. Dashed lines and colour-coded arrowheads indicate the growth fraction; colour-coded arrows indicate the time point at which the labelling index reaches a plateau (TC–TS).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105305&req=5

f2: Cumulative EdU labelling of proliferative and neurogenic APs and BPs.(a, b) Pax6 (magenta), Tbr2 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual AP nuclei indicated by arrows/arrowheads in (a) are shown at higher magnification in (b) (asterisks); white arrowheads, Pax6+/Tbr2−/Tis21-GFP−/EdU− nucleus; white arrows, Pax6+/Tbr2−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Pax6+/Tbr2−/Tis21-GFP+/EdU− nucleus; yellow arrows, Pax6+/Tbr2−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (a) and 10 μm (b). (c, d) Tbr2 (magenta), Tbr1 (blue), Tis21-GFP (green) and EdU (white) staining after cumulative EdU labelling for 9 h. Individual BP nuclei indicated by arrows/arrowheads in (c) are shown at higher magnification in (d) (asterisks); white arrowheads, Tbr2+/Tbr1−/Tis21-GFP−/EdU− nucleus; white arrows, Tbr2+/Tbr1−/Tis21-GFP−/EdU+ nucleus; yellow arrowheads, Tbr2+/Tbr1−/Tis21-GFP+/EdU− nucleus; yellow arrows, Tbr2+/Tbr1−/Tis21-GFP+/EdU+ nucleus. VZ and SVZ are indicated on the left. Scale bars, 50 μm (c) and 10 μm (d). (e, f) Proportion of EdU-labelled NPC nuclei (EdU labelling index) after cumulative EdU labelling for 0.5, 1, 2, 3, 5, 9, 12, 18, 24 and 29 h. The EdU labelling index was separately determined for the various AP (Pax6/Tbr2/GFP/EdU staining as in (a)) and BP (Tbr2/Tbr1/GFP/EdU staining as in (c)) populations, as follows: total APs (e, Tbr2−/Pax6+, blue circles), total BPs (e, Tbr1−/Tbr2+, red squares), proliferative APs (f, Tbr2−/Pax6+/Tis21-GFP−, blue diamonds), BP-genic/neurogenic APs (f, Tbr2−/Pax6+/Tis21-GFP+, purple squares), proliferative BPs (f, Tbr1−/Tbr2+/Tis21-GFP−, red triangles) and neurogenic BPs (f, Tbr1−/Tbr2+/Tis21-GFP+, green crosses). Data are the mean of three 225-μm-wide fields (except for the 1-, 9- and 29-h time points, which are from five, two and one field(s), respectively), each from a different brain and litter; error bars indicate s.e.m. or the range of the two individual values. Dashed lines and colour-coded arrowheads indicate the growth fraction; colour-coded arrows indicate the time point at which the labelling index reaches a plateau (TC–TS).
Mentions: We next used the above information to determine the cell-cycle parameters of Tis21-GFP-negative (Tis21-GFP−) and -positive (Tis21-GFP+) APs and BPs, using cumulative labelling with 5-ethynyl-2-deoxyuridine (EdU). As described in Supplementary Note S2, it was appropriate to use EdU instead of 5-bromo-2-deoxyuridine (BrdU; Supplementary Figs S3 and S4). Cumulative EdU labelling was performed in conjunction with triple immunostaining for either Pax6, Tbr2 plus Tis21-GFP (Fig. 2a,b) or Tbr2, Tbr1 plus Tis21-GFP (Fig. 2c,d) to determine the cell-cycle parameters for APs (Tbr2−/Pax6+) and BPs (Tbr1−/Tbr2+), respectively, and to distinguish between proliferative (Tis21-GFP−) and neurogenic (Tis21-GFP+) NPC sub-populations. As shown in Figure 2e and summarized in Table 1, this revealed that the length of G2+M+G1 (TC–TS), as indicated by the time point at which the EdU labelling index reached the plateau, was longer for the total population of BPs (23.3 h) than APs (14.1 h). In contrast, the proportion of the cell cycle comprising S-phase, as indicated by the intercept of the cumulative EdU labelling curve with the y axis, was smaller for BPs (12%) than for APs (26%). The growth fraction was nearly 100% for both, APs and BPs. Calculation of the length of S-phase (TS) and the total cell cycle (TC)24 yielded values of 5.0 and 19.1 h for APs and 3.2 and 26.5 h for BPs. The present TC data determined by cumulative EdU labelling are very similar to those determined by live imaging in organotypic slice culture720.

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.


Related in: MedlinePlus