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Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain.

Kosodo Y, Suetsugu T, Suda M, Mimori-Kiyosue Y, Toida K, Baba SA, Kimura A, Matsuzaki F - EMBO J. (2011)

Bottom Line: Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms.In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically.Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Kobe, Japan. kosodo@med.kawasaki-m.ac.jp

ABSTRACT
A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

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Related in: MedlinePlus

Tpx2 shows temporal expression and association with microtubules in neural progenitor cells, and loss of Tpx2 function perturbs basal-to-apical nuclear migration. (A) Immunostaining for Tpx2 (a), incorporation of BrdU followed by a 2-h incubation (a′), and the merged view (a″; Tpx2, green; BrdU, magenta) in a cryosection of E14.5 mouse brain tissue. Open arrowheads indicate the Tpx2 and BrdU double-positive cells. Arrows indicate Tpx2 signals outside the nucleus. Note that dividing cells showed strong expression of Tpx2 on their mitotic spindles (white arrowheads, apical mitotic cell; red arrowhead, basal progenitor cell). Bar=10 μm. (B) (a) Schematic showing the cell cycle-dependent translocation of GFP in CCPM-electroporated cells. (b) Co-labelling of E13.5 mouse brain tissue using Tpx2 antibody, CCPM and the merged view (b″; Tpx2, magenta; CCPM, green). The white arrowhead indicates the nucleus of a G1-phase neural progenitor cell, whereas the white arrow indicates the apical process of a G2-phase cell identified by CCPM localization. Bar=10 μm. (C) Expression of GFP (a) or GFP-Tpx2 (b) in neural progenitor cells in E13.5 mouse brain tissue. Note that GFP-Tpx2 localizes to nuclei and apical processes extended in the VZ but not to basal processes. Bar=50 μm. (D) Co-expression of CCPM (a), 6myc-TPX2 (a′) and the merged view (a″; CCPM, green; 6myc-Tpx2, magenta). Red arrowheads in (a′) indicate 6myc-Tpx2 localization at apical processes. Bar=10 μm. (E) HVEM image of GFP-Tpx2 in neural progenitor cells. (a, b) A plasmid encoding GFP-Tpx2 was electroporated into E12.5 mouse brain tissue and incubated for 24 h before dissection. Immunostaining using gold particles was performed on vibratome sections, followed by specimen preparation for HVEM analysis. Note the gold particles localized within the nucleus (a) and on several fibre-like structures in the apical processes (a, b). N, nucleus. Bars=1 μm. (F) Nuclear positions after BrdU incorporation in S-phase followed by a 1-h or 30-min incubation with LacZ miR RNAi as a control (cont., grey dots) or Tpx2 miR RNAi (RNAi, green dots) in E13.5 mouse brain tissue. y-coordinate: distance from apical surface (below 90 μm), EP: NLS-GFP-positive nuclei (electroporated cells), noEP; NLS-GFP-negative nuclei in the same microscopic frame (magenta dots). Black error bars indicate standard error of the mean (s.e.m.). (G) Tracking of basal-to-apical nuclear movement with LacZ miR RNAi as a control (a) or Tpx2 miR RNAi (b) in slice cultures prepared from E13.5 mouse brain tissue. Positions of nuclei relative to the apical surface (y-coordinate) were measured according to their incubation time (x-coordinate). The time point at which nuclei showed the most apical localization was defined as zero. Numbers and colour codes of nuclei are indicated on the right. For (A–E), apical surface is down.
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f3: Tpx2 shows temporal expression and association with microtubules in neural progenitor cells, and loss of Tpx2 function perturbs basal-to-apical nuclear migration. (A) Immunostaining for Tpx2 (a), incorporation of BrdU followed by a 2-h incubation (a′), and the merged view (a″; Tpx2, green; BrdU, magenta) in a cryosection of E14.5 mouse brain tissue. Open arrowheads indicate the Tpx2 and BrdU double-positive cells. Arrows indicate Tpx2 signals outside the nucleus. Note that dividing cells showed strong expression of Tpx2 on their mitotic spindles (white arrowheads, apical mitotic cell; red arrowhead, basal progenitor cell). Bar=10 μm. (B) (a) Schematic showing the cell cycle-dependent translocation of GFP in CCPM-electroporated cells. (b) Co-labelling of E13.5 mouse brain tissue using Tpx2 antibody, CCPM and the merged view (b″; Tpx2, magenta; CCPM, green). The white arrowhead indicates the nucleus of a G1-phase neural progenitor cell, whereas the white arrow indicates the apical process of a G2-phase cell identified by CCPM localization. Bar=10 μm. (C) Expression of GFP (a) or GFP-Tpx2 (b) in neural progenitor cells in E13.5 mouse brain tissue. Note that GFP-Tpx2 localizes to nuclei and apical processes extended in the VZ but not to basal processes. Bar=50 μm. (D) Co-expression of CCPM (a), 6myc-TPX2 (a′) and the merged view (a″; CCPM, green; 6myc-Tpx2, magenta). Red arrowheads in (a′) indicate 6myc-Tpx2 localization at apical processes. Bar=10 μm. (E) HVEM image of GFP-Tpx2 in neural progenitor cells. (a, b) A plasmid encoding GFP-Tpx2 was electroporated into E12.5 mouse brain tissue and incubated for 24 h before dissection. Immunostaining using gold particles was performed on vibratome sections, followed by specimen preparation for HVEM analysis. Note the gold particles localized within the nucleus (a) and on several fibre-like structures in the apical processes (a, b). N, nucleus. Bars=1 μm. (F) Nuclear positions after BrdU incorporation in S-phase followed by a 1-h or 30-min incubation with LacZ miR RNAi as a control (cont., grey dots) or Tpx2 miR RNAi (RNAi, green dots) in E13.5 mouse brain tissue. y-coordinate: distance from apical surface (below 90 μm), EP: NLS-GFP-positive nuclei (electroporated cells), noEP; NLS-GFP-negative nuclei in the same microscopic frame (magenta dots). Black error bars indicate standard error of the mean (s.e.m.). (G) Tracking of basal-to-apical nuclear movement with LacZ miR RNAi as a control (a) or Tpx2 miR RNAi (b) in slice cultures prepared from E13.5 mouse brain tissue. Positions of nuclei relative to the apical surface (y-coordinate) were measured according to their incubation time (x-coordinate). The time point at which nuclei showed the most apical localization was defined as zero. Numbers and colour codes of nuclei are indicated on the right. For (A–E), apical surface is down.

Mentions: The dependence of basal-to-apical migration on both cell-cycle progression (Figure 2) and microtubules (Supplementary Figure S2; Supplementary Movie S2; Tsai et al, 2005; Xie et al, 2007) indicates that microtubule regulation coupled with the cell cycle might control the timing of nuclear migration. Based on reports from other studies (Gruss et al, 2002; Schatz et al, 2003; Gruss and Vernos, 2004), one possible candidate molecule fulfilling this role is Tpx2. Tpx2 is a microtubule-nucleating/bundling protein involved in spindle pole formation around chromosomes dependent on Ran-GTP activity (Gruss and Vernos, 2004). In HeLa cells, Tpx2 protein expression is regulated by the cell cycle; it accumulates in nuclei during S/G2-phase, localizes at the spindle pole during M-phase, and is degraded in early G1-phase (Gruss et al, 2002). We first characterized the protein expression pattern of Tpx2 in neural progenitor cells in the embryonic mouse brain. BrdU labelling to identify the phase of the cell cycle (Takahashi et al, 1992) showed that Tpx2 is expressed in neural progenitor cells in S-, G2- and M-phases (Figure 3A, open arrowheads). We noted that, in addition to the nuclear staining, Tpx2 was found in a fibre-like pattern within VZ cells (Figure 3Aa, arrows). Introduction of cell cycle phase marker (CCPM; Figure 3Ba; Supplementary Movie S3) into neural progenitor cells showed that these fibre-like structures were located in the apical processes only during G2-phase, and were absent in G1-phase (Figure 3Bb; Supplementary Figure S3A), suggesting that Tpx2 functions in the apical processes of the G2-phase neural progenitor cells. When GFP-tagged Tpx2 was expressed in neural progenitor cells, GFP-Tpx2 specifically localized to the nuclei and apical processes of cells in the VZ (Figure 3C). During interphase, GFP-Tpx2 in the apical processes extended toward centrosomes on the apical surface, whereas during M-phase, it was present on mitotic spindles (Supplementary Figure S3B). Co-electroporation of CCPM and 6myc-Tpx2 revealed that the exogenous Tpx2 protein was distributed within the apical processes but never within the basal processes of G2-phase neural progenitor cells (Figure 3D). Ultrastructural analysis using high-voltage electron microscopy (HVEM) showed bundled fibre-like patterns of GFP-Tpx2 reminiscent of microtubule structure within the apical processes (Figure 3E). The microtubule bundling activity of Tpx2 had been previously shown in a purified in vitro system (Schatz et al, 2003), and when taken together, our results strongly suggest that Tpx2 is associated with microtubules in the apical processes of neural progenitor cells. Furthermore, even though both the basal and apical processes of these cells contain large amount of microtubules, the localization of Tpx2 is restricted to the apical processes, suggesting that its association with microtubules is not promiscuous (Supplementary Figure S3C).


Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain.

Kosodo Y, Suetsugu T, Suda M, Mimori-Kiyosue Y, Toida K, Baba SA, Kimura A, Matsuzaki F - EMBO J. (2011)

Tpx2 shows temporal expression and association with microtubules in neural progenitor cells, and loss of Tpx2 function perturbs basal-to-apical nuclear migration. (A) Immunostaining for Tpx2 (a), incorporation of BrdU followed by a 2-h incubation (a′), and the merged view (a″; Tpx2, green; BrdU, magenta) in a cryosection of E14.5 mouse brain tissue. Open arrowheads indicate the Tpx2 and BrdU double-positive cells. Arrows indicate Tpx2 signals outside the nucleus. Note that dividing cells showed strong expression of Tpx2 on their mitotic spindles (white arrowheads, apical mitotic cell; red arrowhead, basal progenitor cell). Bar=10 μm. (B) (a) Schematic showing the cell cycle-dependent translocation of GFP in CCPM-electroporated cells. (b) Co-labelling of E13.5 mouse brain tissue using Tpx2 antibody, CCPM and the merged view (b″; Tpx2, magenta; CCPM, green). The white arrowhead indicates the nucleus of a G1-phase neural progenitor cell, whereas the white arrow indicates the apical process of a G2-phase cell identified by CCPM localization. Bar=10 μm. (C) Expression of GFP (a) or GFP-Tpx2 (b) in neural progenitor cells in E13.5 mouse brain tissue. Note that GFP-Tpx2 localizes to nuclei and apical processes extended in the VZ but not to basal processes. Bar=50 μm. (D) Co-expression of CCPM (a), 6myc-TPX2 (a′) and the merged view (a″; CCPM, green; 6myc-Tpx2, magenta). Red arrowheads in (a′) indicate 6myc-Tpx2 localization at apical processes. Bar=10 μm. (E) HVEM image of GFP-Tpx2 in neural progenitor cells. (a, b) A plasmid encoding GFP-Tpx2 was electroporated into E12.5 mouse brain tissue and incubated for 24 h before dissection. Immunostaining using gold particles was performed on vibratome sections, followed by specimen preparation for HVEM analysis. Note the gold particles localized within the nucleus (a) and on several fibre-like structures in the apical processes (a, b). N, nucleus. Bars=1 μm. (F) Nuclear positions after BrdU incorporation in S-phase followed by a 1-h or 30-min incubation with LacZ miR RNAi as a control (cont., grey dots) or Tpx2 miR RNAi (RNAi, green dots) in E13.5 mouse brain tissue. y-coordinate: distance from apical surface (below 90 μm), EP: NLS-GFP-positive nuclei (electroporated cells), noEP; NLS-GFP-negative nuclei in the same microscopic frame (magenta dots). Black error bars indicate standard error of the mean (s.e.m.). (G) Tracking of basal-to-apical nuclear movement with LacZ miR RNAi as a control (a) or Tpx2 miR RNAi (b) in slice cultures prepared from E13.5 mouse brain tissue. Positions of nuclei relative to the apical surface (y-coordinate) were measured according to their incubation time (x-coordinate). The time point at which nuclei showed the most apical localization was defined as zero. Numbers and colour codes of nuclei are indicated on the right. For (A–E), apical surface is down.
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f3: Tpx2 shows temporal expression and association with microtubules in neural progenitor cells, and loss of Tpx2 function perturbs basal-to-apical nuclear migration. (A) Immunostaining for Tpx2 (a), incorporation of BrdU followed by a 2-h incubation (a′), and the merged view (a″; Tpx2, green; BrdU, magenta) in a cryosection of E14.5 mouse brain tissue. Open arrowheads indicate the Tpx2 and BrdU double-positive cells. Arrows indicate Tpx2 signals outside the nucleus. Note that dividing cells showed strong expression of Tpx2 on their mitotic spindles (white arrowheads, apical mitotic cell; red arrowhead, basal progenitor cell). Bar=10 μm. (B) (a) Schematic showing the cell cycle-dependent translocation of GFP in CCPM-electroporated cells. (b) Co-labelling of E13.5 mouse brain tissue using Tpx2 antibody, CCPM and the merged view (b″; Tpx2, magenta; CCPM, green). The white arrowhead indicates the nucleus of a G1-phase neural progenitor cell, whereas the white arrow indicates the apical process of a G2-phase cell identified by CCPM localization. Bar=10 μm. (C) Expression of GFP (a) or GFP-Tpx2 (b) in neural progenitor cells in E13.5 mouse brain tissue. Note that GFP-Tpx2 localizes to nuclei and apical processes extended in the VZ but not to basal processes. Bar=50 μm. (D) Co-expression of CCPM (a), 6myc-TPX2 (a′) and the merged view (a″; CCPM, green; 6myc-Tpx2, magenta). Red arrowheads in (a′) indicate 6myc-Tpx2 localization at apical processes. Bar=10 μm. (E) HVEM image of GFP-Tpx2 in neural progenitor cells. (a, b) A plasmid encoding GFP-Tpx2 was electroporated into E12.5 mouse brain tissue and incubated for 24 h before dissection. Immunostaining using gold particles was performed on vibratome sections, followed by specimen preparation for HVEM analysis. Note the gold particles localized within the nucleus (a) and on several fibre-like structures in the apical processes (a, b). N, nucleus. Bars=1 μm. (F) Nuclear positions after BrdU incorporation in S-phase followed by a 1-h or 30-min incubation with LacZ miR RNAi as a control (cont., grey dots) or Tpx2 miR RNAi (RNAi, green dots) in E13.5 mouse brain tissue. y-coordinate: distance from apical surface (below 90 μm), EP: NLS-GFP-positive nuclei (electroporated cells), noEP; NLS-GFP-negative nuclei in the same microscopic frame (magenta dots). Black error bars indicate standard error of the mean (s.e.m.). (G) Tracking of basal-to-apical nuclear movement with LacZ miR RNAi as a control (a) or Tpx2 miR RNAi (b) in slice cultures prepared from E13.5 mouse brain tissue. Positions of nuclei relative to the apical surface (y-coordinate) were measured according to their incubation time (x-coordinate). The time point at which nuclei showed the most apical localization was defined as zero. Numbers and colour codes of nuclei are indicated on the right. For (A–E), apical surface is down.
Mentions: The dependence of basal-to-apical migration on both cell-cycle progression (Figure 2) and microtubules (Supplementary Figure S2; Supplementary Movie S2; Tsai et al, 2005; Xie et al, 2007) indicates that microtubule regulation coupled with the cell cycle might control the timing of nuclear migration. Based on reports from other studies (Gruss et al, 2002; Schatz et al, 2003; Gruss and Vernos, 2004), one possible candidate molecule fulfilling this role is Tpx2. Tpx2 is a microtubule-nucleating/bundling protein involved in spindle pole formation around chromosomes dependent on Ran-GTP activity (Gruss and Vernos, 2004). In HeLa cells, Tpx2 protein expression is regulated by the cell cycle; it accumulates in nuclei during S/G2-phase, localizes at the spindle pole during M-phase, and is degraded in early G1-phase (Gruss et al, 2002). We first characterized the protein expression pattern of Tpx2 in neural progenitor cells in the embryonic mouse brain. BrdU labelling to identify the phase of the cell cycle (Takahashi et al, 1992) showed that Tpx2 is expressed in neural progenitor cells in S-, G2- and M-phases (Figure 3A, open arrowheads). We noted that, in addition to the nuclear staining, Tpx2 was found in a fibre-like pattern within VZ cells (Figure 3Aa, arrows). Introduction of cell cycle phase marker (CCPM; Figure 3Ba; Supplementary Movie S3) into neural progenitor cells showed that these fibre-like structures were located in the apical processes only during G2-phase, and were absent in G1-phase (Figure 3Bb; Supplementary Figure S3A), suggesting that Tpx2 functions in the apical processes of the G2-phase neural progenitor cells. When GFP-tagged Tpx2 was expressed in neural progenitor cells, GFP-Tpx2 specifically localized to the nuclei and apical processes of cells in the VZ (Figure 3C). During interphase, GFP-Tpx2 in the apical processes extended toward centrosomes on the apical surface, whereas during M-phase, it was present on mitotic spindles (Supplementary Figure S3B). Co-electroporation of CCPM and 6myc-Tpx2 revealed that the exogenous Tpx2 protein was distributed within the apical processes but never within the basal processes of G2-phase neural progenitor cells (Figure 3D). Ultrastructural analysis using high-voltage electron microscopy (HVEM) showed bundled fibre-like patterns of GFP-Tpx2 reminiscent of microtubule structure within the apical processes (Figure 3E). The microtubule bundling activity of Tpx2 had been previously shown in a purified in vitro system (Schatz et al, 2003), and when taken together, our results strongly suggest that Tpx2 is associated with microtubules in the apical processes of neural progenitor cells. Furthermore, even though both the basal and apical processes of these cells contain large amount of microtubules, the localization of Tpx2 is restricted to the apical processes, suggesting that its association with microtubules is not promiscuous (Supplementary Figure S3C).

Bottom Line: Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms.In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically.Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Kobe, Japan. kosodo@med.kawasaki-m.ac.jp

ABSTRACT
A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

Show MeSH
Related in: MedlinePlus