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First published online 18 March 2008
doi: 10.1242/jcs.025064

Journal of Cell Science 121, 1204-1212 (2008)
Published by The Company of Biologists 2008
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Cell-cycle-specific nestin expression coordinates with morphological changes in embryonic cortical neural progenitors

Takehiko Sunabori1,2,3, Akinori Tokunaga1,3, Takeharu Nagai4,5, Kazunobu Sawamoto1,2, Masaru Okabe6, Atsushi Miyawaki4, Yumi Matsuzaki1, Takaki Miyata7 and Hideyuki Okano1,3,*

1 Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
2 Bridgestone Laboratory of Developmental and Regenerative Neurobiology, Keio University School of Medicine, Tokyo 160-8582, Japan
3 Solution Oriented Research for Evolutional Science and Technology (SORST), Japan Science and Technology Agency (JST), Saitama 332-0012, Japan
4 Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
5 Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Hokkaido 060-0812, Japan
6 Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
7 Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan


Figure 1
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  		Fig. 1. E/nestin:dVenus fluorescence intensity is correlated with Nes mRNA expression. (A) Structure of the E/nestin:dVenus transgene. (B) Emission spectra of Venus (green) and EGFP (red). (C,D) On an identical section from an E14 E/nestin:dVenus-EGFP double transgenic embryo, no dVenus-expressing cells showed immunoreactivity against the anti-β-tubulin III antibody, although some EGFP-expressing cells did (arrowheads). VZ, ventricular zone; IZ, intermediate zone. (E) Representative FACS profile of cerebral-wall-derived cells from an E14.5 E/nestin:dVenus transgenic embryo. (F) Quantitative data showing the percentage of E/nestin:dVenus negative (F1) and positive (F2) cells immunostained with nestin, β-tubulin III and GFP antibodies. (G) To compare the E/nestin:dVenus fluorescence intensity with the Nes mRNA expression level, real-time quantitative PCR was performed. dVenus-positive cells were subdivided into two fractions based on their fluorescence intensity: high (F4) and low (F3). The Nes mRNA expression level of these two fractions (F4 and F3), as well as of the dVenus-negative cells (F1), showed a positive correlation with the fluorescence intensity. Data are means ± s.e.m. Scale bars: 500 µm (C), 25 µm (D).

 

Figure 2
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  		Fig. 2. Cell-cycle-dependent expression of Nes. (A) Schematic diagram of the timing used to administer thymidine analogues to label cells in each cell-cycle phase (see Materials and Methods for details). (B-D) IdU (red) was injected for 0.5 hour (B), 3 hours (C) and 14.5 hours (D) to label cells in S, G2-M and G1 phases, respectively. BrdU (purple) was injected 30 minutes before sacrifice to label cells in S phase. dVenus (green) was expressed in many of the cells in G1 and S phase (arrowheads), but cells in the G2-M phase were mostly dVenus negative (arrows). (E) Ratio of IdU-positive cells that expressed dVenus to the total number of IdU-positive cells, counted for each cell-cycle phase. Cells in G1 and S phase expressed the highest levels of dVenus, whereas its expression levels were much lower during G2-M phase. Scale bar: 25 µm (B-D). Data represent the mean ± s.e.m.

 

Figure 3
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  		Fig. 3. The relationship between Nes expression levels and the 3D morphology of neural progenitor cells. (A) VZ cells were labeled with DiI (red) from the pial surface and observed for 4 hours after mitosis. The radial fiber, which became extremely thin during mitosis, was inherited by one of the daughter cells (arrowhead i). Four hours after mitosis, both daughter cells expressed dVenus. Within this period, the cell that inherited the process thickened its fiber and extended 20 µm (arrowhead i), whereas the cell that did not inherit the fiber began to elaborate its own process (arrowhead ii). (B) During G2 phase, the VZ progenitor cells possessed a mature fiber, and each nucleus migrated toward the ventricular surface. dVenus was not expressed in the cells during this phase (arrowhead). (C) Cells in M phase, labeled with phospho-histone H3 (PH3), were dVenus-negative both near the ventricular surface (arrowheads) and in the subventricular zone (arrow). The asterisk indicates the pial surface. Scale bar: 25 µm (A-C).

 

Figure 4
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  		Fig. 4. The expression of Nes is reduced by the decreased affinity of Brn2 phosphorylated on Ser362 for the Nes core enhancer during G2-M. (A) Immunostaining showed that only a subset of SOX1-SOX3 and Brn2 immunopositive cells expressed dVenus in the E14.5 cerebral cortex. (B) Flag-tagged Brn2- and Brn2-S362A-expressing 293T cells were synchronized at the G1, S and G2-M phases and labeled with [32P]orthophosphate. The low level of Brn2 phosphorylation at G1 and S (lane 1, 2) increased during the G2-M phase (lane 3). However, the phosphorylation level of Brn2 S362A at G2-M (lane 5) was the same as in the G1 phase (lane 4). (C) SOX2, Brn2 and Brn2 S362D were used to transfect cells either alone, or as a SOX-POU combination, with a luciferase-encoding reporter plasmid carrying an octamerized nestin core enhancer sequence (8xNes30). Together, SOX2 and Brn2 synergistically activated the 8xNes30 reporter. By contrast, when Brn2 was replaced with Brn2 S362D, the activation was markedly lower. Data represent means ± s.e.m. (D) EMSA showing the lower binding affinity of Brn2 S362D (lane 3) compared with the wild-type Brn2 (lane 2) for the Nes30 probe (black arrow). The binding specificity was confirmed by a super shift assay (black arrowhead; lanes 4, 5). No binding reaction was seen with the lysate of untransfected 293T cells (lane 1). Open arrowhead indicates the free probe. (E) Western blot showing the Brn2 and Brn2 S362D input used for the EMSA. No significant difference was seen between the stability of Brn2 and Brn2 S362D when normalized to {alpha}-tubulin expression (*). Scale bar: 25 µm.

 

Figure 5
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  		Fig. 5. A schematic diagram of Nes expression according to the cell-cycle-dependent morphological changes of neural progenitor cells in the developing cerebral cortex. (A) A schematic diagram of the stage-dependent thickening of the mouse cerebral wall based on Takahashi et al. (Takahashi et al., 1993Go). Cortical progenitor cells elongate as development proceeds from an early `neuroepithelial cell' to a mid-embryonic `radial glial cell' stage. The expression of Nes (green) was strong during the neurogenic stage, which accompanied obvious cerebral wall thickening and the prolonged G1 phase. (B) Cell-cycle-dependent morphological changes of mid-embryonic progenitor cells. At the beginning of the G1 phase, each progenitor cell generated at the ventricular surface is either connected to the pial surface (cell i) or lacking a pial process (cell ii). The daughter cell that inherited the pial process (cell i) elongates its process within a single cell cycle to match the cerebral wall thickening. The other daughter cell (cell ii) elaborates a new pial process, mainly during G1, and adopts the bipolar radial glial morphology by S phase. During mitosis, the cell body rounds up and the process becomes extremely thin. A model for the lineage-restriction of the progenitor cell is omitted for simplification. The expression of Nes (green) was observed concomitant with the elongation of the radial process during the G1 to S phases. At G2-M, Nes expression declined because of the phosphorylation of its upstream regulator class III POU protein, allowing the cells to undergo mitosis. Note that the duration of each cell cycle phase illustrated (G1, S, and G2-M) is according to those determined at E14 by Takahashi et al. (Takahashi et al., 1995Go). CP, cortical plate; IMZ, intermediate zone; VZ, ventricular zone.

 

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© The Company of Biologists Ltd 2008