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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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Cell-cycle arrest in S-phase perturbs apical-to-basal nuclear movement of neural progenitor cells in G1-phase. (A) Assessment of drug conditions that inhibit S-phase of neural progenitor cells in slice cultures prepared from E13.5 mouse brain tissue. (a–c) Immunostaining of cryosections prepared from brain slices after drug treatment. Concentrations of HU are indicated in the panels. Green, phosphorylated Histone H3 (mitotic cells); red, ZO-1 (apical surface). Bar=50 μm. (d) M-phase neural progenitor cells in cultured slices after drug treatment. The average number of mitotic cells per 100 μm of apical surface under several conditions are indicated. Note that HU resulted in a concentration-dependent reduction of M-phase neural progenitor cells. FU, 5-fluorouracil. (B) Tracking of nuclear movements in brain slice cultures with HU treatment. Nuclei of neural progenitor cells were marked with NLS-GFP, and their distances from the apical surface (y-coordinate) were plotted versus their incubation time (x-coordinate). Red arrowheads indicate the time point when control vehicle (a) or 1 mM HU (b) was added to the medium (6 h after incubation started). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicates daughter cells derived from cell division at the apical surface). (C) Apical-to-basal nuclear movements following cell division at the apical surface during 8 h of incubation after adding (a) control vehicle, (b) 0.5 mM HU or (c) 1 mM HU. Colour codes are explained above. (d) Average velocities of nuclei are shown in (a–c) (20–22 cases in each condition). **P<0.005, ***P<0.001, t-test. Error bars indicate s.e.m. (D) Effect of HU treatment on microbead movement induced from the apical surface. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from time-lapse images. Six hours after adding microbeads to the brain slice cultures, control vehicle (a) or 1 mM HU (b) was added to the medium (indicated as time point 0). Tracked movements of 10 microbeads in each condition are displayed. (E) Effect of co-existing G1-arrested cells on the basally oriented nuclear movement of non-arrested cells. After in utero electroporation of plasmids of p18Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before fixing the embryos. With this procedure, CldU single-labelled cells were identified as having been in S-phase 2–5 h before fixation. During this time window, CldU single-labelled cells reach the apical surface, therefore their distribution pattern indicates nuclear position of late M-phase or G1-phase cells. Nuclei of p18Ink4c plasmid-negative cells were identified by the absence of NLS-GFP signals, and their position from the apical surface was measured. Histogram shows the comparison of the GFP-negative CldU single-labelled nuclear positions of six electroporated brains from three independent experiments (total number of measured nuclei; control n=550, p18Ink4c n=594).
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f6: Cell-cycle arrest in S-phase perturbs apical-to-basal nuclear movement of neural progenitor cells in G1-phase. (A) Assessment of drug conditions that inhibit S-phase of neural progenitor cells in slice cultures prepared from E13.5 mouse brain tissue. (a–c) Immunostaining of cryosections prepared from brain slices after drug treatment. Concentrations of HU are indicated in the panels. Green, phosphorylated Histone H3 (mitotic cells); red, ZO-1 (apical surface). Bar=50 μm. (d) M-phase neural progenitor cells in cultured slices after drug treatment. The average number of mitotic cells per 100 μm of apical surface under several conditions are indicated. Note that HU resulted in a concentration-dependent reduction of M-phase neural progenitor cells. FU, 5-fluorouracil. (B) Tracking of nuclear movements in brain slice cultures with HU treatment. Nuclei of neural progenitor cells were marked with NLS-GFP, and their distances from the apical surface (y-coordinate) were plotted versus their incubation time (x-coordinate). Red arrowheads indicate the time point when control vehicle (a) or 1 mM HU (b) was added to the medium (6 h after incubation started). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicates daughter cells derived from cell division at the apical surface). (C) Apical-to-basal nuclear movements following cell division at the apical surface during 8 h of incubation after adding (a) control vehicle, (b) 0.5 mM HU or (c) 1 mM HU. Colour codes are explained above. (d) Average velocities of nuclei are shown in (a–c) (20–22 cases in each condition). **P<0.005, ***P<0.001, t-test. Error bars indicate s.e.m. (D) Effect of HU treatment on microbead movement induced from the apical surface. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from time-lapse images. Six hours after adding microbeads to the brain slice cultures, control vehicle (a) or 1 mM HU (b) was added to the medium (indicated as time point 0). Tracked movements of 10 microbeads in each condition are displayed. (E) Effect of co-existing G1-arrested cells on the basally oriented nuclear movement of non-arrested cells. After in utero electroporation of plasmids of p18Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before fixing the embryos. With this procedure, CldU single-labelled cells were identified as having been in S-phase 2–5 h before fixation. During this time window, CldU single-labelled cells reach the apical surface, therefore their distribution pattern indicates nuclear position of late M-phase or G1-phase cells. Nuclei of p18Ink4c plasmid-negative cells were identified by the absence of NLS-GFP signals, and their position from the apical surface was measured. Histogram shows the comparison of the GFP-negative CldU single-labelled nuclear positions of six electroporated brains from three independent experiments (total number of measured nuclei; control n=550, p18Ink4c n=594).

Mentions: Microbead translocation in the brain slice suggests that G1-nuclei are driven in the apical-to-basal direction by a non-autonomous mechanism. However, such a mechanism is not immediately apparent. One possibility is that active basal-to-apical migration of nuclei increases the nuclear density high on the apical side of the VZ. The resultant close packing of nuclei may crowd out free nuclei that have completed mitosis, so that they are pushed further away from the ventricular surface. If this is the case, apical-to-basal migration should be affected by the acute perturbation of basal-to-apical migration of nuclei in G2-phase. To test this hypothesis, we arrested the cell cycle at S-phase by drug treatment and performed time-lapse observations of migrating nuclei. We first searched for an appropriate inhibitor to arrest the cell cycle of neural progenitor cells in the mouse brain slice culture in S-phase. Hydroxyurea (HU), which selectively inhibits ribonucleoside diphosphate reductase (Wright et al, 1990), leads to a dose-dependent reduction in the number of mitotic cells in the VZ (Figure 6A) without inducing apoptosis below 1 mM (Supplementary Figure S7A). As previously reported in chick neuroepithelium (Murciano et al, 2002), we confirmed that HU treatment results in an accumulation of S-phase neural progenitor cells by measuring the rate of BrdU incorporation in nuclei immediately after washing out the HU (Supplementary Figure S7B). The results indicated that the G1–S transition was not impaired by HU treatment. We tracked nuclear movement under these conditions in slice cultures to see the effect of HU administration (Figure 6B; Supplementary Movies S9 and S10). In the presence of HU, the number of apically migrating nuclei was dramatically decreased and S-phase nuclei accumulated in the basal region of the VZ. This result was expected, as HU arrests progenitor cells at S-phase, preventing entry into G2-phase. Time-lapse observations revealed that apical-to-basal migration was also quickly perturbed by HU treatment (Figure 6B and C). Under this condition, the average velocity of nuclei moving in the basal direction was significantly decreased (Figure 6Cd). We did note a few rapidly moving unidentified nuclei (3/42 cases with HU, Figure 6C); however, the majority were impeded. This acute delay in apical-to-basal migration is most likely due to the lack of a decrease in basal nuclear density, and of an increase in apical nuclear density, both of which are simultaneously caused by individual nuclei migrating apically in the normal situation. To confirm that the perturbation of basally directed nuclear movement is not due to unexpected effects of HU on G1-phase cells, but instead due to the physical displacement effect, we tested whether microbead translocation (Figure 5) is also perturbed by the same drug treatment. Indeed, most fluorescent beads incorporated from the apical surface translocated shorter distances after treatment with 1 mM HU than in the control (Figure 6D). To further corroborate the nuclear displacement effects, we arrested the cell cycle of a population of cells at G1-phase by introducing p18Ink4c plasmid (see Figure 2), and examined the distribution of pulse-labelled nuclei (by two thymidine analogues: CldU and IdU, see the figure legend), that have not incorporated p18Ink4c plasmid (i.e. not affected by the cell-cycle arrest). When p18Ink4c plasmid was introduced to the surrounding cells, the distribution of CldU single-labelled nuclei of p18Ink4c plasmid-negative cells showed a notable accumulation close to the apical surface (Figure 6E), indicating that apical-to-basal nuclear migration is perturbed. Based on the results above, we conclude that the apical-to-basal nuclear migration that occurs in G1-phase is subject to a displacement or crowding-out effect of incoming nuclei migrating in the opposite direction after exiting S-phase.


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)

Cell-cycle arrest in S-phase perturbs apical-to-basal nuclear movement of neural progenitor cells in G1-phase. (A) Assessment of drug conditions that inhibit S-phase of neural progenitor cells in slice cultures prepared from E13.5 mouse brain tissue. (a–c) Immunostaining of cryosections prepared from brain slices after drug treatment. Concentrations of HU are indicated in the panels. Green, phosphorylated Histone H3 (mitotic cells); red, ZO-1 (apical surface). Bar=50 μm. (d) M-phase neural progenitor cells in cultured slices after drug treatment. The average number of mitotic cells per 100 μm of apical surface under several conditions are indicated. Note that HU resulted in a concentration-dependent reduction of M-phase neural progenitor cells. FU, 5-fluorouracil. (B) Tracking of nuclear movements in brain slice cultures with HU treatment. Nuclei of neural progenitor cells were marked with NLS-GFP, and their distances from the apical surface (y-coordinate) were plotted versus their incubation time (x-coordinate). Red arrowheads indicate the time point when control vehicle (a) or 1 mM HU (b) was added to the medium (6 h after incubation started). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicates daughter cells derived from cell division at the apical surface). (C) Apical-to-basal nuclear movements following cell division at the apical surface during 8 h of incubation after adding (a) control vehicle, (b) 0.5 mM HU or (c) 1 mM HU. Colour codes are explained above. (d) Average velocities of nuclei are shown in (a–c) (20–22 cases in each condition). **P<0.005, ***P<0.001, t-test. Error bars indicate s.e.m. (D) Effect of HU treatment on microbead movement induced from the apical surface. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from time-lapse images. Six hours after adding microbeads to the brain slice cultures, control vehicle (a) or 1 mM HU (b) was added to the medium (indicated as time point 0). Tracked movements of 10 microbeads in each condition are displayed. (E) Effect of co-existing G1-arrested cells on the basally oriented nuclear movement of non-arrested cells. After in utero electroporation of plasmids of p18Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before fixing the embryos. With this procedure, CldU single-labelled cells were identified as having been in S-phase 2–5 h before fixation. During this time window, CldU single-labelled cells reach the apical surface, therefore their distribution pattern indicates nuclear position of late M-phase or G1-phase cells. Nuclei of p18Ink4c plasmid-negative cells were identified by the absence of NLS-GFP signals, and their position from the apical surface was measured. Histogram shows the comparison of the GFP-negative CldU single-labelled nuclear positions of six electroporated brains from three independent experiments (total number of measured nuclei; control n=550, p18Ink4c n=594).
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f6: Cell-cycle arrest in S-phase perturbs apical-to-basal nuclear movement of neural progenitor cells in G1-phase. (A) Assessment of drug conditions that inhibit S-phase of neural progenitor cells in slice cultures prepared from E13.5 mouse brain tissue. (a–c) Immunostaining of cryosections prepared from brain slices after drug treatment. Concentrations of HU are indicated in the panels. Green, phosphorylated Histone H3 (mitotic cells); red, ZO-1 (apical surface). Bar=50 μm. (d) M-phase neural progenitor cells in cultured slices after drug treatment. The average number of mitotic cells per 100 μm of apical surface under several conditions are indicated. Note that HU resulted in a concentration-dependent reduction of M-phase neural progenitor cells. FU, 5-fluorouracil. (B) Tracking of nuclear movements in brain slice cultures with HU treatment. Nuclei of neural progenitor cells were marked with NLS-GFP, and their distances from the apical surface (y-coordinate) were plotted versus their incubation time (x-coordinate). Red arrowheads indicate the time point when control vehicle (a) or 1 mM HU (b) was added to the medium (6 h after incubation started). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicates daughter cells derived from cell division at the apical surface). (C) Apical-to-basal nuclear movements following cell division at the apical surface during 8 h of incubation after adding (a) control vehicle, (b) 0.5 mM HU or (c) 1 mM HU. Colour codes are explained above. (d) Average velocities of nuclei are shown in (a–c) (20–22 cases in each condition). **P<0.005, ***P<0.001, t-test. Error bars indicate s.e.m. (D) Effect of HU treatment on microbead movement induced from the apical surface. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from time-lapse images. Six hours after adding microbeads to the brain slice cultures, control vehicle (a) or 1 mM HU (b) was added to the medium (indicated as time point 0). Tracked movements of 10 microbeads in each condition are displayed. (E) Effect of co-existing G1-arrested cells on the basally oriented nuclear movement of non-arrested cells. After in utero electroporation of plasmids of p18Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before fixing the embryos. With this procedure, CldU single-labelled cells were identified as having been in S-phase 2–5 h before fixation. During this time window, CldU single-labelled cells reach the apical surface, therefore their distribution pattern indicates nuclear position of late M-phase or G1-phase cells. Nuclei of p18Ink4c plasmid-negative cells were identified by the absence of NLS-GFP signals, and their position from the apical surface was measured. Histogram shows the comparison of the GFP-negative CldU single-labelled nuclear positions of six electroporated brains from three independent experiments (total number of measured nuclei; control n=550, p18Ink4c n=594).
Mentions: Microbead translocation in the brain slice suggests that G1-nuclei are driven in the apical-to-basal direction by a non-autonomous mechanism. However, such a mechanism is not immediately apparent. One possibility is that active basal-to-apical migration of nuclei increases the nuclear density high on the apical side of the VZ. The resultant close packing of nuclei may crowd out free nuclei that have completed mitosis, so that they are pushed further away from the ventricular surface. If this is the case, apical-to-basal migration should be affected by the acute perturbation of basal-to-apical migration of nuclei in G2-phase. To test this hypothesis, we arrested the cell cycle at S-phase by drug treatment and performed time-lapse observations of migrating nuclei. We first searched for an appropriate inhibitor to arrest the cell cycle of neural progenitor cells in the mouse brain slice culture in S-phase. Hydroxyurea (HU), which selectively inhibits ribonucleoside diphosphate reductase (Wright et al, 1990), leads to a dose-dependent reduction in the number of mitotic cells in the VZ (Figure 6A) without inducing apoptosis below 1 mM (Supplementary Figure S7A). As previously reported in chick neuroepithelium (Murciano et al, 2002), we confirmed that HU treatment results in an accumulation of S-phase neural progenitor cells by measuring the rate of BrdU incorporation in nuclei immediately after washing out the HU (Supplementary Figure S7B). The results indicated that the G1–S transition was not impaired by HU treatment. We tracked nuclear movement under these conditions in slice cultures to see the effect of HU administration (Figure 6B; Supplementary Movies S9 and S10). In the presence of HU, the number of apically migrating nuclei was dramatically decreased and S-phase nuclei accumulated in the basal region of the VZ. This result was expected, as HU arrests progenitor cells at S-phase, preventing entry into G2-phase. Time-lapse observations revealed that apical-to-basal migration was also quickly perturbed by HU treatment (Figure 6B and C). Under this condition, the average velocity of nuclei moving in the basal direction was significantly decreased (Figure 6Cd). We did note a few rapidly moving unidentified nuclei (3/42 cases with HU, Figure 6C); however, the majority were impeded. This acute delay in apical-to-basal migration is most likely due to the lack of a decrease in basal nuclear density, and of an increase in apical nuclear density, both of which are simultaneously caused by individual nuclei migrating apically in the normal situation. To confirm that the perturbation of basally directed nuclear movement is not due to unexpected effects of HU on G1-phase cells, but instead due to the physical displacement effect, we tested whether microbead translocation (Figure 5) is also perturbed by the same drug treatment. Indeed, most fluorescent beads incorporated from the apical surface translocated shorter distances after treatment with 1 mM HU than in the control (Figure 6D). To further corroborate the nuclear displacement effects, we arrested the cell cycle of a population of cells at G1-phase by introducing p18Ink4c plasmid (see Figure 2), and examined the distribution of pulse-labelled nuclei (by two thymidine analogues: CldU and IdU, see the figure legend), that have not incorporated p18Ink4c plasmid (i.e. not affected by the cell-cycle arrest). When p18Ink4c plasmid was introduced to the surrounding cells, the distribution of CldU single-labelled nuclei of p18Ink4c plasmid-negative cells showed a notable accumulation close to the apical surface (Figure 6E), indicating that apical-to-basal nuclear migration is perturbed. Based on the results above, we conclude that the apical-to-basal nuclear migration that occurs in G1-phase is subject to a displacement or crowding-out effect of incoming nuclei migrating in the opposite direction after exiting S-phase.

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