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

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.

Figure 1

Quantitative tracking of nuclear movement of cortical neural progenitor cells in embryonic mouse brain slice cultures. (A) Schematic model of INM of neural progenitor cells. Nuclei in the VZ, the closest tissue layer to the ventral surface of the developing brain, show an oscillatory movement along the apical–basal epithelial axis that is associated with the phase of the cell cycle (see Introduction). The colour code of cell-cycle phases is indicated on the right. (B) Representative movement of a nucleus undergoing INM. Nuclei of neural progenitor cells were labelled by NLS-GFP, and their movements in slice cultures prepared from E13.5 mouse brains were tracked by time-lapse microscopy. Using the tracking software, positions of nuclei from 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. Phases of the cell cycle, estimated from previous reports, are indicated below (see Results). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicate daughter cells derived from cell division at the apical surface).

Figure 2

Arresting the cell cycle in G1-phase by introduction of p18^Ink4c induces accumulation of nuclei in the basal region of the VZ. (A) Immunostaining of E14.5 mouse brain treated with p18^Ink4c to arrest the cell cycle in G1-phase with a Ki67 antibody (proliferative marker, red). (a) Contralateral side. (b, b′) E13.5 mouse lateral cortex in which p18^Ink4c (b) and NLS-GFP (b′; green) were co-electroporated and incubated for 24 h. Bar=50 μm. (B) Expression of GFP (green) with electroporation of either control vector (a) or p18^Ink4c (b, b′) into E10.5 mouse telencephalon followed by 18 h of whole-embryo culture. Immunostaining using a p18^Ink4c antibody was performed (b′; red). Bar=10 μm. (C) Positions of NLS-GFP-expressing nuclei with control vector (a) or p18^Ink4c (b) introduced by electroporation into E13.5 mouse cortex followed by a 24-h incubation. Co-staining using a Tuj1 antibody (neuronal marker, red) and DAPI (DNA, blue) was performed. Bar=10 μm. (D) Positions of NLS-GFP-expressing nuclei relative to the apical surface with control vector (a) or p18^Ink4c (b). Sums of numbers (N, x-coordinate) counted in 15 electroporated brain sections (nuclei from each section are indicated by white or dotted boxes) are shown according to their distance from the apical surface (y-coordinate). For (A–C), apical surface is down.

Figure 3

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.

Figure 4

Tpx2 alters microtubule organization in the apical process of G2-phase neural progenitor cells. (A) Images of cell cycle phase marker (CCPM; a) and EB3-tagRFP (a′) in living brain slice cultures. CCPM and EB3-tagRFP were co-electroporated into neural progenitor cells in E12.5 mouse brain, and fluorescent signals from two separate wavelengths were acquired from living tissue slices prepared from E13.5 brain. Note that CCPM localization inside the nucleus indicates that the cell is in G1-phase (filled arrowhead in a and a″), whereas its localization peripheral to the nucleus indicates that the cell is in G2-phase (open arrowhead in a and a″). Arrows in a′ and a″ indicate organizations of microtubules in the apical process identified by EB3-tagRFP. (B) Examples of microtubule organization in each cell-cycle phase combined with knockdown of Tpx2 functions. Images were acquired as described in (A). LacZ miR RNAi (control, a, b) or Tpx2 miR RNAi (c, d) were co-electroporated together with CCPM and EB3-tagRFP. Cell-cycle phases identified by the localization of CCPM are indicated in the panel. Arrowheads and arrows are as described in (A). (C) Quantitation of EB3-tagRFP in the apical process. (a) Scanning of EB3-tagRFP signal in the apical process. The pixel intensity of tagRFP fluorescence in the region of the apical process close to the nucleus (dotted rectangle, 5 μm in length) was measured using MetaMorph software. The double arrow indicates the direction of scanning. (b) Relative intensity of EB3-tagRFP fluorescence in each cell-cycle phase combined with knockdown of Tpx2 functions. The pixel intensity of tagRFP fluorescence in each condition (colour codes are indicated in the panel) was quantified as described in (a). Signals were normalized as follows: after subtracting the background level from the raw digitized value of pixel intensity, the remaining value was divided by the sum of all the background-corrected values to calculate percentages (y-coordinate). Error bars indicate s.e.m. Bars in (A–C)=10 μm. (D) Ultrastructural analysis of Tpx2 function on microtubule organization. LacZ miR RNAi (control, a) or Tpx2 miR RNAi (b) were co-electroporated together with CCPM into neural progenitor cells in E13.5 mouse brain, and a slice culture was prepared. After the fixation and permeabilization, pre-embed immunolabelling (Toida et al, 2000) was performed on the brain slice using GFP antibody and 3,3′-diaminobenzidine (DAB) reaction to identify the apical process of G2-phase cells. After embedding, 50-nm thick ultrathin sections parallel to the apical surface were prepared to observe the cross-sections of the apical process (10–15 μm away from the apical surface). Asterisk in (b) show DAB-negative cell, its microtubules are seen as small dots. Note that DAB precipitation around microtubules is distinct in DAB-positive cells (arrows in a, b). Bar=500 nm. (c, d) Number of microtubules in a single (c) or unit area (0.1 μm²) (d) of the cross-sections of DAB-positive apical process in control or Tpx2 knocked-down situation (eight cases in each condition). ^***P<0.001, t-test (in d). Error bars indicate s.e.m. For (A–C), apical surface is down.

Figure 5

Unidirectional translocation of microbeads from the apical surface toward the basal region of the VZ. (A) Schematic of the method used to incorporate fluorescent microbeads (diameters are ∼2 μm) into embryonic mouse brain tissue using magnetic activity, followed by tracking of their movement in slice cultures using time-lapse microscopy. (B) Images of fluorescent beads in slice cultures (a, b; red) incorporated into E13.5 mouse brain tissue. The white dashed line indicates the apical surface. Beads were aligned at the apical surface at the starting time point (a). Several beads had detached from their original position after 24 h of incubation (b). Bar=50 μm. (C) Tracking of microbeads in brain slice cultures. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from the time-lapse images. Movements of nine microbeads acquired from three independent brain slices are displayed (slice numbers (1–3) and beads (A–D) are indicated in the graph). (D) Immunostaining of mouse brain sections from cultured tissue slices after microbead incorporation (a, b; red). Antibody for proliferating cell nuclear antigen (PCNA) stains proliferative cells (a′; green), whereas Tuj1 stains post-mitotic neurons (a, a′; magenta). Note that fluorescent microbeads were observed only in the proliferative zone, the VZ (white arrowheads). Yellow dashed lines indicate the apical surface. Bar=10 μm. (E) Comparison of nuclear and microbead tracks in brain tissue slices. Plane positions of nuclei marked with NLS-GFP (a) or incorporated microbeads (b) were measured along the apical surface (x-coordinate) and apical–basal axis (y-coordinate). Slice numbers (1–3), nuclei and beads (A–D) are indicated in each graph.

Figure 6

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 p18^Ink4c 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 p18^Ink4c 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, p18^Ink4c n=594).

Figure 7

Computational modelling of INM combined with in vivo experiments. (A) (a) Scheme of nuclear movement posited in the model. The nucleus goes to the centre of gravity of an area that is unoccupied by neighbouring nuclei within the SR. r, radius of nucleus; SR, searching radius. (b) One of the frames from the sequential movie of modelling analyses visualized for interpretations (see Supplementary Movie S11). Some nuclei are highlighted by colour codes according to their phase in the cell cycle: blue, G1/S; green, G2; orange, M. Nuclei can cross the border of the lateral and basal sides but not that of the apical side. See details in Supplementary data. (B) Movement of 12 individual nuclei acquired from the computational model without (a) or with (b) basal-to-apical nuclear movement during G2-phase. x-coordinate: time, y-coordinate: distance from the apical surface. Note that the patterns of apical-to-basal movement and the points reached before entering G2-phase differ significantly among nuclei (b). (C) (a) Effect of changing parameters in the computational model. Average distances from the apical surface of 200 nuclei and their phase in the cell cycle in the simulation were plotted. Length of G1-phase in E11.5 (3.2 h) and E14.5 (9.3 h) and whether cell division at M-phase is occurring (+ or −) are indicated. (b) Relative positions of nuclei that exited S-phase between 3–6 h before the end of the simulation using the cell-cycle parameters for E11.5 (grey) or E14.5 (green). (D) (a) Incorporation of CldU (magenta) and IdU (green) into the nuclei of neural progenitor cells of mouse brains 6 and 3 h before fixation, respectively. Blue arrow: the presence of a CldU single-labelled nucleus implies that it exited S-phase between 6 and 3 h before fixation. Distance from the apical surface (yellow dashed line) was measured (d). Bar=50 μm. (b) Positions of nuclei that exited S-phase between 6 and 3 h before fixation in E11.5 (grey) and E14.5 (green) embryonic mouse brains. (E) Histogram view of the results of the double-labelling experiment acquired from the computational model (a, see (C)) or in vivo mouse brains (b, see (D)). Proportions of nuclei (x-coordinate) at specific distances from the apical surface (y-coordinate, rounded off to a two-digit number).