1 Neurology, Massachusetts General Hospital, Boston, MA, USA, 2 Pediatrics, Keio University School of Medicine, Tokyo, Japan and 3 Neuroscience & Cell Biology, UMDNJ-RWJ Medical School, Piscataway, NJ, USA
Address correspondence to Verne S. Caviness, Department of Neurology, Massachusetts General Hospital, 55 Fruit Street, VBK-901, Boston, MA 02114, USA. Email: caviness{at}helix.mgh.harvard.edu
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Abstract |
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Key Words: cell cycle cell specification neurogenesis p27Kip1 tet-system
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Introduction |
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Materials and Methods |
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Double transgenic (DT) mice, in which p27Kip1 can be overexpressed selectively and electively in the neuroepithelium of the embryonic brain, were produced by mating the hemizygous tetOp27Kip1/ and PnestinrtTA/ FVB lines described previously (Mitsuhashi et al., 2001). The plug date was defined as embryonic day 0 (E0). Wild-type (WT) littermates were used as controls. Each experiment was initiated by induction of p27Kip1 overexpression in response to orally administered doxycycline hydrochloride (dox; Sigma) to pregnant dams at a dose of 25 µg/g body wt, twice daily from E12 to E14 (Fig. 1). All of the experimental procedures were in full compliance with institutional guidelines and the NIH Guide for the Care and Use of Laboratory Animals.
Induction of p27Kip1 Overexpression and S-phase Labeling
Each experiment was initiated by induction of p27Kip1 expression by oral dox administration to pregnant dams (Fig. 1A). The following four iododeoxyuridinebromodeoxyuridine (IdU and BrdU, respectively) double S-phase labeling paradigms were employed.
Cell Cycle Kinetics (Fig. 1B)
A single i.p. injection of IdU (Sigma; 50 µg/g) was administered to pregnant dams carrying E14 mice at 07:00 h. It was followed at 09:00 h by a single injection of BrdU (Sigma; 50 µg/g). Mice were killed at 09:30 h (Hayes and Nowakowski, 2000).
Neuronogenetic Interval (Fig. 1C)
BrdU was administered to pregnant dams carrying E16 mice as a single i.p. injection at either 09:00 or 21:00 h on E16. Offspring were killed on postnatal day 4 (P4) and the distribution of BrdU-labeled cells was mapped in the cerebral cortex with respect to cortical cytoarchitectonic fields (Fig. 2).
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IdU was administered as a single i.p. injection to pregnant dams carrying E14 mice at 07:00 h. This was followed by sequential i.p. administrations of BrdU every 3 h from 09:00 to 24:00 h corresponding to an interval longer than TC TS (birth hour method: Takahashi et al., 1996a,b
). This design identifies a cohort of cells that were in S-phase between 07:00 and 09:00 h and that exited the cell cycle following the S-phase. The cohort is labeled only with IdU (Fig. 3C). Since we count only IdU-only labeled cells, the cells that reenter S-phase (rather than exiting the cell cycle) disappear, as they will be double-labeled with BrdU and IdU (Fig. 3C).
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The design is identical to that for NQ estimation (Fig. 1D) except that the 2 h cohort of IdU-only labeled cells is examined in the cerebral cortex on P21 (Fig. 4D,E) (Takahashi et al., 1999).
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P4 and P21 mice were anesthetized (Ketamine, 50 µg/g; Ketalar, Abbott; Xylazine 10 µg/g; Rompun, Bayer; i.p.) and perfused through the heart with 4% paraformaldehyde in phosphate buffer, pH 7.2. Brains were removed, embedded in paraffin wax. E14 mice were removed by hysterotomy from anesthetized dams and decapitated. The entire heads were embedded in paraffin wax. The postnatal brains and embryonic heads were sectioned at a thickness of 4 µm in the coronal plane. The sections were processed for IdUBrdU double immunohistochemistry as described below. Tail (postnatal) or trunk (embryo) samples were collected for genotyping from anesthetized mice prior to tissue fixation.
IdUBrdU Immunohistochemistry
Paraffin-embedded sections were cleared in Histoclear (National Diagnostic) and xylene (Fisher Scientific), rehydrated in graded ethanol and PBS. The sections were immersed in 5% acetic acid overnight, then washed with distilled water, and treated with 0.2% trypsin (37°C, 20 min) and 2 N HCl (30 min). The sections from E14 mice were microwaved in a solution of 0.01 M sodium citrate, pH 6.2, for 15 min and not trypsinized, as trypsinization adversely affected the integrity of the sections. Non-specific antibody reaction was blocked with 1.5% normal horse serum in PBS (30 min). Sections were incubated with mouse monoclonal antibody Br3 (Caltag Lab., 0.025% in PBS, 1.5% normal horse serum and 0.5% Tween-20) for 30 min, biotinylated anti-mouse IgG (0.5% in PBS) for 45 min and then with Vector ABC-peroxidase solution (ABC Peroxidase Elite kit, Vector) for 60 min. The sections were reacted with diaminobenzidine (DAB, 0.05%, Sigma) and H2O2 (0.01%) for 8 15 min, rinsed with PBS, immersed in 5% acetic acid for 30 min and washed with distilled water. The sections were then incubated with mouse monoclonal antibody IU4 (Caltag Lab., 0.025% in PBS and 0.5% Tween-20) for 30 min, biotinylated anti-mouse IgG (0.5% in PBS) for 45 min and then with vector ABC alkaline phosphatase solution (ABC-Alkaline phosphatase kit, Vector) for 60 min. The sections were reacted with Alkaline Phosphatase Substrate (Vector Blue-Alkaline Phosphatase Substrate Kit III, Vector) for 15 min. The sections were rinsed with distilled water and coverslipped with Crystal Mount (Biomedia).
TUNEL Histochemistry
Paraformaldehyde-fixed, paraffin-embedded, 4 µm thick, coronal sections were processed for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) according to the manufacturer's instructions (ApopTag kit, Intergen Purchase). The sections were counterstained with 0.1% aqueous basic fuchsin. TUNEL-positive cells were counted in the cortical gray matter (layer VI to the pial surface) of fields 1 and 40 (Verney et al., 2000).
Analysis of IdUBrdU-labeled Cells
The monoclonal antibody IU4 detects both IdU and BrdU. IU4-labeled cells stain blue due to the alkaline phosphatase reaction product. Br3 detects only BrdU. Br3-labeled cells stain brown due to the DAB reaction product (Hayes and Nowakowski, 2000). Thus, although the IdUBrdU labeling paradigms (Fig. 2B,D,E) yield three types of labeled cells IdU-only, BrdU-only, and BrdU and IdU double-labeled cells only the IdU-only labeled cells (blue, solid arrowhead in Fig. 3C), which were in S-phase at the time of the IdU injection and exited the S-phase by the time of the BrdU injection, can be considered to be specifically identifiable. Both the BrdU-only labeled and IdUBrdU-double labeled cells (blue/brown, open arrowhead in Fig. 3C) would have been in S-phase at the time of the BrdU injection (Nowakowski et al., 1989
; Takahashi et al., 1999
; Hayes and Nowakowski, 2000
). Due to the antibody specificity, we attach no significance to the double labeled versus BrdU-only labeled population. Note, specifically, that our assay depends only on identifying the IdU-only labeled cells. There are also cells not labeled with either IdU or BrdU. Those cells were not in S-phase when effective labeling concentrations of either tracer were available (Fig. 3AC).
The analyses were performed at two locations along the mediallateral axis of the VZ, namely in the medial cortical zone (MCZ) and the lateral cortical zone (LCZ; Fig. 3A,B). We counted all three types of labeled cells in the MCZ and the LCZ of E14 mice (Fig. 3A,B) within a sector that was 100 µm in its mediallateral dimension and 4 µm (corresponding to section thickness) in its rostralcaudal dimension (Fig. 3C). The radial dimension of the sector was divided into bins. Each bin is 10 µm high. The bins were numbered 1, 2, 3, etc., from the ventricular margin outward. Following the IdU-only labeled cell counts, the coverslips were removed and the sections were stained with 0.1% aqueous basic fuchsin so that all cells in the VZ could be counted. The number of unlabeled cells was obtained by subtracting IdU and/or BrdU labeled cells from all cells.
Labeled cells were counted also in the medially located field 1 and laterally located field 40 (Caviness, 1975) in P21 cerebral cortex (Fig. 4D,E). For convenience and consistency with our previous work (Takahashi et al., 1999
; Caviness et al., 2003
), we divided the cortical gray matter into granularsupragranular (SG: layers IVII/III) and infragranular (IG: layers VIV) layers. In order to correlate judgments of the architectonic landmarks of the two cortical fields with the various labeling patterns in P21 mice, we stained the IdUBrdU-labeled sections with basic fuchsin as described above (Fig. 4B,C). In these sections, we identified the border between layers V (internal pyramidal cell layer) and IV (internal granular cell layer) based on cell morphology (large pyramidal cells in layer V and smaller granular cells in IV) and cell packing density (relatively low packing density in layer V compared to that in layer IV). The entire gray matter between the pial surface and white matter/layer VI border was divided into bins as in the E14 studies. We divided the cortical gray matter into SG and IG layers (Fig. 4B,C). We confirmed the registration of the IdU-only labeled cells to IG and SG layers, retrospectively by re-registering the bins with cortical layers (Fig. 4D, Table 4). We also counted the total number of nuclei (i.e. all nuclei) in IG and SG (Table 3).
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Analysis of Cell Cycle Kinetics
To measure the length of the cell cycle (TC) and its constituent phases [TG1, TS (the length of S-phase) and TG2+M (combined lengths of G2- and M-phases)], three types of labeled cells in the labeling paradigm of Figure 2B were counted in the VZ: IdU-only labeled cells (NI), BrdU-only and BrdU and IdU double-labeled cells (NB) and all cells (NT). The number of unlabeled cells (N0) was calculated by N0 = NT NI NB (Table 1). Kinetic parameters are derived according to a modification of the double-labeling algorithm formulated by Hayes and Nowakowski (2000). We determined that TG2+M was
2 h, as the majority of M-phase cells (99%) were labeled with IdU-only during the 2 h labeling period (Takahashi et al., 1995
) in both WT and DT mice. The growth fraction (GF; Takahashi et al., 1995
) was
1.0, as cumulative BrdU administration over 15 h labeled all cells in the VZ of WT and DT mice (data not shown). Since the number of cells after M-phase is doubled by mitosis, the number of unlabeled cells that contributes to the cell cycle is N0/2. Similarly the total number of cells that contributes to the cell cycle is NT N0/2. With these considerations, TC and TS are estimated from the number of cells of each labeling group according to the following equations.
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The BrdU-labeled sections of P4 brains were photographed using a Nikon Eclipse E400 microscope and a digital camera (SPOT RT Slider Camera, RT230-2, Diagnostic Instruments). The images were processed by SPOT Advanced v.3.5 software (Diagnostic Instruments) and Adobe Photoshop 7.0 (Adobe). The position of the BrdU-labeled cells in the cortical gray matter was superimposed on the outlines of the sections (Fig. 2A,B). The BrdU labeling pattern reconstructed from the serial sections was superimposed on a surface view of the cortical area map (Fig. 2C,D).
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Results |
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The mating between the hemizygous tetOp27Kip1/ and PnestinrtTA/ mice was as frequently associated with vaginal plugs as mating among WT mice, although the incidence of successful impregnation was lower in the former. Once pregnancy was established, however, the course of gestation was not altered by the hemizygous condition and parturition occurred on E19, just as for the WT mice. In addition, the number of live born offspring per litter was not different in the hemizygous matings compared to the WT matings. Finally, the DT, hemizygote and WT genotype ratios from tetOp27Kip1/ x PnestinrtTA/ mating approximated the expected Mendelian ratio of 1:2:1.
Cell Cycle Kinetics
We used a double S-phase labeling method to estimate cell cycle parameters in the VZ of the cerebral wall in E14 DT and WT littermates exposed to dox from E12 onwards (Fig. 1B). We chose E14 for analysis because it is the time when the laminar fate of neurons shifts from layer V to IV in the somastosensory cortex of normal mouse (Takahashi et al., 1999) (Fig. 1A). The analyses were performed at MCZ and LCZ (Fig. 3A,B). The progenitors in the LCZ are developmentally in advance of those in the MCZ by at least 24 h, with respect to neuronogenetic schedule (Takahashi et al., 1995
, 1996a
,b
, 1999
). Thus, this single experiment is the equivalent of examining the same area of cortex at both E14 and E15, and the analyses in the two zones provide a method to determine if the effects of p27Kip1 overexpression are dependent upon the stage of maturation of the progenitors.
The number of IdU- and BrdU-labeled (i.e. BrdU+ or IdU+/BrdU+) cells was recorded in the MCZ and LCZ of DT and WT littermates (Table 1) to estimate the length of cell cycle (TC) and its phases [TG1, TS (the duration of S-phase) and TG2+M (combined lengths of G2- and M-phases)] according to a method described previously (Hayes and Nowakowski, 2000). The cell cycle parameters did not differ between DT and WT mice either in the MCZ or the LCZ (Table 2). Thus, overexpression of p27Kip1 from E12 to E14 does not alter either the total length of cell cycle or the lengths of its constituent phases on E14.
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Since the cell cycle parameters did not change in the DT mice, the total number of integer cell cycles executed over the duration of the neuronogenetic interval itself would not be expected to change. We tested that prediction next. Neuronogenesis is completed throughout the neocortical PVE of the mouse early on E17 (Caviness, 1982; Takahashi et al., 1995
; Miyama et al., 1997
). The process of termination of neuronogenesis follows a transverse neurogenetic gradient (Bayer and Altman, 1991
) such that wave fronts of initiation and termination of neuronal production advance along the rostrolateral to caudomedial axis of the hemisphere (Miyama et al., 1997
). The position of the termination wave front is particularly clear at the end of the neuronogenetic period because it marks the border between an area of the PVE that continues to produce neurons and an area in which the PVE has involuted and is no longer producing cells. If p27Kip1 overexpression altered the neuronogenetic interval, the position of the termination wave front at the end of the neuronogenetic period would be displaced in the DT mice compared to the WT littermates, and the degree of displacement would be commensurate with the extent of alteration in the neuronogenetic interval.
We visualized the position of the termination wave front in DT and WT mice at the end of the neuronogenetic period by using a BrdU labeling method (Fig. 1C). We administered BrdU at 09:00 or 21:00 h on E16 and observed the distribution of BrdU-positive cells in the neocortical gray matter in serial coronal sections of the brain on P4 (see examples in Fig. 2A,B). The position of BrdU-positive cells in the cortical gray matter was recorded with respect to the section outlines (Fig. 2A,B). The lateral extent of the spread of the BrdU label (the termination wave front) for representative cases from the 09:00 and 21:00 injections is indicated by arrows at the level of the somatosensory (barrel field) cortex in Figure 2A,B. The termination wave front is located more medially following the 9:00 injection than the 21:00 injection at this coronal level (compare Fig. 2A to 2B), as predicted by the neurogenetic gradient. We reconstructed the termination wave front from the serial sections and superimposed it on a surface view of the cortical area map (Fig. 2C,D). In both WT and DT mice, following the 09:00 h BrdU injection, the front of labeled cells was located a short distance from the rhinal fissure along a plane that passed through insular field 14 laterally and the lateral temporal and perirhinal field 36 posteriorly (Fig. 2C), in accordance with the predicted labeling pattern (Caviness, 1975; Miyama et al., 1997
). Following the 21:00 h BrdU injection, the termination wave front was located further into the lateral parietal fields 3a and 40 medially and the lateral occipital field 18a posteriorly in both the genotypes (Fig. 2D). Thus, we found that the tangential and radial distributions of the E16 BrdU labeled cells in the P4 neocortex were indistinguishable between DT (Fig. 2A,B) and WT littermates (data not shown). Therefore, within the limits of resolution of the methods used here, neither the duration of the neuronogenetic interval nor the slope of the transverse neurogenetic gradient were altered by the overexpression of p27Kip1.
Probability of Cell Cycle Exit
Each of the two daughter cells resulting from a round of cell division has a quantifiable probability of re-entry into or exit from the cell cycle. These probability values are designated as P (for reentry) and Q (for exit) (Takahashi et al., 1996a,b
). P + Q is always 1.0, as these two fates are complementary and cell death in the PVE is small, i.e. <1% per cell cycle (Haydar et al., 2000b
; Cai et al., 2002
). P and Q are calculated based on an estimation of the number of cells in the P and Q fractions (NP and NQ, respectively). This is accomplished by labeling a cohort of cells undergoing S-phase over an experimentally defined interval and following the cohort as it executes G2-, M- and G1-phases so as to distinguish cells that exit the cell cycle from those that re-enter it (Takahashi et al., 1996a
,b
). We calculated NQ and NP in E14 DT and WT embryos exposed to dox from E12 to E14 using the IdUBrdU labeling protocol. We calculated NQ using the labeling protocol shown in Figure 1D. In this protocol cells stained blue (labeled only with IdU) are the NQ cells (Fig. 3AC) (Takahashi et al., 1996a
,b
). We calculated NP+Q by doubling the value of NI (Table 1) obtained using the labeling protocol shown in Figure 1B. Q was calculated using the formula Q = NQ/(NP + NQ). We found that there was a statistically significant increase (P-value = 0.03, t-test) in Q in MCZ of DT mice compared to WT littermates (Table 2), whereas the measures of Q in the LCZ of DT and WT littermates were not different.
We also recorded the pattern of distribution of the NQ cells in the VZ and the intermediate zone (IZ) as they exited the VZ and migrated toward the marginal zones. The distribution of these cells represented the distance of migration 6 h after exiting cell cycle. The cells which exited cell cycle have left the VZ and migrated across the IZ to traverse
90% (MCZ) and
70% (LCZ) of the height of the cerebral wall at the time of measurement (Takahashi et al., 1996a
,b
). Therefore the pattern of distribution represented the migratory rate of these cells, which was indistinguishable in DT and WT littermates (Fig. 3D,E). Moreover, the architectonic appearance of the developing cortex in DT animals was normal, including cortical plate and subplate and the course of the sagittal stratum subjacent to the cortical strata. There was no suggestion of architectonic anomalies such as are seen in reeler (Caviness, 1982
) and other cortical mutants (Walsh, 2000
; Ohshima et al., 2001
; Hammond et al., 2004
) where there is disorder of migration and postmigration mechanisms of laminar assembly. Therefore, patterns of cell exit from the VZ and migration across the IZ as well as postmigration mechanisms of laminar assembly are unperturbed by p27Kip1 overexpression. The analysis of postnatal migration and laminar assembly will be considered later in the Discussion.
Cytoarchitecture of the Cerebral Cortex at Postnatal Day 21
We examined the histology of the cerebral cortex in the DT and WT littermates on P21. There were no differences between WT and DT mice in the gross appearance of the brain as seen in a dorsal view (Fig. 4A). In histological sections, however, there are clear differences in the cortical width. Thus, the neocortex is distinctly thinner in the DT, especially in field 1 (Fig. 4B) albeit less in field 40 (Fig. 4C). This is a quantitative difference only. In particular, the stratification and laminar cytoarchitectonic patterns are similar in the DT and WT mice in neocortical fields 1 and 40 (Fig. 4B,C). Moreover, regionally distinguishing architectonic features e.g. the barrel patterns of area 3, and the full array of sublaminar patterns through the parietal, frontal temporal, occipital and medial hemispheric fields were also preserved in the DT mice (data not shown). Thus, the p27Kip1 overexpression from E12 to E14 did not produce gross malformations or architectonic pattern abnormalities of the cerebral cortex or other brain regions at P21. We address the quantitative issues related to cortical thickness in the next section.
Cortical Thickness and Neuronal Number at P21
Overexpression of p27Kip1 was associated with a modest reduction in the radial thickness and the total number of neurons in the cortex in field 1 in the DT mice ( 8% and
10%, respectively) (Table 3). This was due entirely to a more substantial reduction in the thickness and number of cells of the SG layers (
24% and
18%, respectively). There was no detectable difference in the thickness of the IG layers of field 1. There was no detectable difference in either the full cortical thickness or cell numbers of field 40 overall or that of either the SG or IG layers in that field.
This contrast in the response to p27Kip1 overexpression between medial and lateral localized cortical fields and of SG with respect to IG layers within field 1 is predicted from a model formalized earlier (Caviness et al., 2000, 2003
). It will be considered further in the Discussion.
Laminar Fates of Cells Born on E14
We next examined if the laminar fates of cells that exited the cell cycle (NQ cells) on E14 were different between the DT and WT littermates on P21, using a modification of the double-S-phase labeling method (Figs 1E, 4D,E) that was used to estimate NQ on E14 (Fig. 1D). In this method, the E14 NQ cells can be recognized in the P21 cortex as blue cells (Fig. 4D,E). These cells appear to be neocortical projection neurons based on the morphology of the somata (data not shown) and also based on previous reports that the output of the neocortical VZ on E14 is virtually exclusively projection neurons (Takahashi et al., 1995; Qian et al., 2000; Malatesta et al., 2003). It is possible that some of the NQ cells are interneurons. However, the interneurons constitute only
1525% of the total number of neocortical neurons in rodents (Ren et al., 1992
; Beaulieu, 1993
) and are unlikely to introduce significant bias in our data. Therefore, we consider the E14 NQ cells to be projection neurons.
We recorded the number and radial distribution of the NQ cells in neocortical fields 1 and 40 in WT and DT mice (field 1; Fig. 4E). First, we measured the total number of NQ cells in the gray matter in fields 1 and 40. We found that the total number increased by 52% in field 1 (destination of cells originating in the MCZ; Takahashi et al., 1999
) and
43% in field 40 (destination of cells originating in the LCZ; Takahashi et al., 1999
) in the DT mice relative to the WT littermates (Table 4). The differences were statistically significant in both the fields. The increase in NQ cells in field 1 of the DT mice was consistent with the increases in NQ and Q in the MCZ of DT mice at E14 (Table 2). However, the increase in NQ cells in field 40 was not paired with an increase in NQ or Q in the LCZ of DT mice at E14. Plausibly the discrepancy here is attributable to the variability of measurement in the relatively small numbers of DT and WT littermates recovered for the E14 studies.
Next, we examined the distribution of the E14 NQ cells in the SG and IG layers. We found that in the WT mice 22% of the NQ cells were distributed to SG layers in field 1 and
56% in field 40 (Table 4), reflecting the maturity differences produced by the down gradient position of field 1 relative to field 40 along the transverse neurogenetic gradient. In the DT mice, by contrast, nearly 42% of the NQ cells were found in SG layers in field 1 and 65% in field 40. Thus, there was an increase in the proportion of the NQ cells of the cohort that had been directed to SG layers in the DT mice in both fields 1 and 40 compared to the WT mice (Fig. 4E). This corresponded to a 94% increase in the proportion of NQ cells directed to SG layers in field 1 of the DT mice compared to the WT mice. In field 40, by contrast, although 65% of the NQ cells were directed to the SG layers in the DT mice, this represented only a 20% increase over the WT mice (Table 4).
These findings indicate that on average in the DT mice an NQ cell produced on E14 in the MCZ has a 94% greater chance of residing in SG layers and a NQ cell from the LCZ a 20% greater chance of residing in the SG layers compared to corresponding cells in the WT (Table 4). We encountered substantial variability among the litters in the relative distribution of the NQ cells, in keeping with the expected variation in relative maturity among the litters (Theiler, 1972; Takahashi et al., 1999
). We exploited this by correlating the percentage of NQ cells directed to SG layers in DT versus WT littermate pairs in fields 1 and 40 (Fig. 5A). The broken line (with 45° slope; Fig. 5A) denotes the no effect line, representing a hypothetical situation, in which DT and WT littermates have identical values (i.e. p27Kip1 overexpression has no effect). However, the slope of the regression line obtained with the actual data is significantly (t-test, P < 0.01) lower than that of the no effect line, indicating that the percentage of cells directed to the SG layers was greater in DT mice compared to the WT littermates. Thus, it is evident that the field 1 values diverge from the no effect line to a greater extent than the field 40 values, reflecting differences in the maturational state of these two areas (Miyama et al., 1997
). This is consistent with the findings from E14 where NQ and Q were significantly higher in the DT mice in the MCZ (precursor of field 1) and not in the LCZ (precursor of field 40) (Table 2). Thus, overexpression of p27Kip1 from E12 to E14 not only shifted the destination of cells produced on E14 towards more superficial layers of the neocortex but also did so in a developmentally regulated manner. That is, there is progressively smaller effect in more mature regions of the cortex (at the time of p27Kip1 induction) as the transverse neurogenetic gradient shifts to formation of principally SG neurons.
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P4 is the time of maximum apoptotic cell death in the developing mouse neocortex, especially in fields 1 and 40 (Verney et al., 2000). No significant difference was detected between DT and WT littermates in the numerical density of TUNEL+ profiles either in field 1 (mean ± SEM, DT 4.36 x 103 ± 1.46 x 103, WT 3.66 x 103 ± 0.51 x 103 cells/unit area, t-test, P = 0.661) or field 40 (mean ± SEM, DT 2.46 x 103 ± 0.92 x 103, WT 3.38 x 103 ± 0.55 x 103 cells/unit area, t-test, P = 0.418). There was also no difference in the laminar distribution of TUNEL+ profiles between WT and DT littermates in either cortical field (data not shown).
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Discussion |
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The capacity of p27Kip1 to increase Q is developmentally regulated, i.e. strongly apparent in the developmentally early MCZ but not detectable in the developmentally late LCZ. The increase in Q is dissociated from the kinetic operation of the cell cycle in that TG1 (and therefore TC) and the overall neuronogenetic interval remain unchanged. This means that the number of cell cycles in the neuronogenetic interval remains fixed at 11 just as in the normal animal. This also means that the path of ascent of Q as a function of cell cycle sequence through the neuronogenetic interval is altered. This path increases non-linearly from 0.0 (before the first neurons are born) to 1.0 with the final cycle when the last cell division occurs (Takahashi et al., 1996a,b
; Nowakowski et al., 2002
). In the DT animal the rate of ascent with cycle is increased over the interval of p27Kip1 overexpression and must therefore be somewhat slowed subsequently. Correspondingly the ascent of Q in the p27Kip1/ mouse must rise abnormally slowly initially but then accelerate with respect to WT in the terminal cycles (Goto et al., 2005
). It is to be noted in this regard that variations in cell cycle parameters, in contrast to variations in Q, will have little effect upon cell production in the neocortical PVE. Thus, output per cycle is an exponential function of Q and total output is simply the cumulative output of all cycles (Takahashi et al., 2000
). Variations in cycle duration may affect linearly the output in time but will have no effect upon output per cycle or total output for a series of cycles. As an example in point, in the Emx2/ mutant there is a profound reduction in neuronal production but cycle durations are unaltered (Mallamaci et al., 2000
). Q, by default the affected parameter, has not been measured. Plausibly the same will hold for Pax6/ and other mutants which are similarly associated with massive limitations of neuronal production (O'Leary and Nakagawa, 2002
; Grove and Fukuchi-Shimogori, 2003
; Shin et al., 2004
).
Finally, we have observed that the effect of p27Kip1 overexpression upon Q is dissociated from post-proliferative histogenetic mechanisms involved in migration and postmigration histogenetic mechanisms of cortical pattern formation and from postmigratory histogenetic cell death. These processes appear to proceed normally in all respects in the E14 DT mouse embryo. Moreover, in confirmation, the stratification and laminar cytoarchitectonic patterns (such as barrel patterns of area 3) are preserved completely in the P21 animal. Recently p27Kip1 has been found to play a role as modulator of cell motility in cultured fibroblasts, a role mediated by RhoA activation (Besson et al., 2004). However, in the present study, p27Kip1 overexpression in the DT mice would have returned to near normal levels by E16, prior to the onset of robust neocortical neuronal migration. Our previous study (Mitsuhashi et al., 2001
) showed that p27Kip1 mRNA levels began to rise 6 h after a single dose of dox, reached 300-fold of baseline expression at 12 h and returned to essentially baseline expression levels 48 h after the dox dose. Therefore, it is unlikely that p27Kip1 overexpression affects migration in this experiment model.
p27Kip1, Q and Neuronal Laminar Destination
The size of the 2 h (cells stained blue) cohort arising on E14 and distributed within fields 1 and 40 at P21 is larger in DT than WT animals. We attribute this to an increase in Q, occurring in response to p27 Kip1 overexpression. We note, moreover, that there is also a substantial increase in the proportion of the cohort that is distributed to SG layers in DT P21 animals. Because TG1 and therefore TC are unaltered at E14 in the DT embryos, the cohort in the P21 cortex must have arisen from the same cell cycle in the 11 cycle sequence in WT and DT animals. However, the composition of this cohort has been altered such that an increased proportion of its cells is destined for SG layers in both fields 1 and 40 though to a larger degree in field 1. For reasons cited above, this phenomenon is not an artifact or a disturbance of migration, postmigration mechanisms of laminar assembly or abnormal patterns of cell death. It cannot be simply incidental to the E14 cohort representing an increased fraction of the total numbers of cells formed on E14 and subsequently in that the architectonic features of these layers are normal. The patterning behavior of the SG cells in DT is indistinguishable from those in WT. Thus, it is the IGSG fate specification profile of the E14 cohort that is changed. In the DT animals, where cells are specified with the characteristics of IG or SG cells, their histogenetic behavior is appropriate to that specification.
Thus, p27Kip1 overexpression is found here to be associated with an alteration in the proportionate laminar destination of cells with respect to the cell cycle number of origin. However, the cardinal insight here is that the proportionate laminar destination of cells is not altered with respect to the value of Q of the cell cycle of origin. In other words, of four possible counting methods embryonic age, cell cycle number, the length of G1, and Q (the probability of exiting the cell cycle) it is the last that is apparently the determinant for laminar position. The evidence for this assertion is based upon the interpretation of the data presented here in the context of previously published data from the WT CD1 strain mouse (Takahashi et al., 1996a,b
, 1999
), in which the relationship between Q and the percentage of the cohort that is destined to be SG cells was first determined (percentage SG cells; Fig. 5B). The percentage of SG cells from the E14 cohort and measured at P21 approximate closely a sigmoidal curve (Fig. 5B; solid line; R2 = 0.99). This suggests a tight regulatory linkage between Q and laminar destination. The data from the present study illustrating the effect of p27Kip1 overexpression also approximates closely a sigmoidal curve (R2 = 0.96). This is represented as a broken line since the y-axis values of its left and right tails would be 0% and 100%, respectively (Takahashi et al., 1996a
,b
, 1999
). The curve for the present transgenic mouse line is displaced to the left, presumably reflecting both differences in the strains and in the methods used, which are recognized to be differentially sensitive to leading and trailing edges of the S phase labeled cohort (Hayes and Nowakowski, 2000
). Thus, the proportions of neurons fated by specification for SG layer positions under conditions of p27Kip1 overexpression are also correlated to Q. The most striking feature of Figure 5A is the effect that the maturity of the neocortex has on the shift produced by the upregulation of p27Kip1. The less mature cortex is more dramatically affected. This is reasonable because the less mature cortex in WT would be producing fewer cells fated to become SG, and, thus, a shift towards SG production in DT would be more dramatic.
These findings imply that mechanisms that regulate Q are coordinate with those that regulate specification of laminar destination and operate before the histogenetic events of laminar assembly and cell differentiation (Fig. 6). The expression of Tis21, an antimitogen, appears to be selectively sensitive to the transition between specification and readiness to exit the cycle. Its profile of expression approximates the measured profile of advance of Q with cell cycle (Iacopetti et al., 1999; Calegari and Huttner, 2003
; Haubensak et al., 2004
; Kosodo et al., 2004
). These investigators propose that over the course of the G1 phase of the cell cycle there is cumulative synthesis of a substance, as yet unidentified, that coordinately increases Q and the expression of Tis21 (Calegari and Huttner, 2003
). Our data are in accord with this model in that they identify p27Kip1 as a substance that drives Q upward as its levels increase. Moreover, we also showed that the linkage between Q and laminar destination is driven by p27Kip1 overexpression. For the present it is premature to suggest that p27Kip1 or substances regulating its synthesis are the exclusive determinants of the advance of Q. The linkage of the p27Kip1 drive of Q may be downstream of the proliferative drive originating with the FGFR1 receptor (Shin et al., 2004
). It must be upstream of the graded concentrations of transcription factors known to be involved in neuron specification (Grove and Fukuchi-Shimogori, 2003
). The actual linkages between Q and regulation of these concentration gradients are as yet unknown.
|
The present observation linking neocortical laminar fate specification to p27Kip1 has precedent in other systems. For example, under optimum growth conditions in vitro, O-2A oligodendrocyte progenitor cells withdraw from cell cycle and differentiate into oligodendroglial cells after a set number of cycles. As cycles proceed there are rising levels of p27Kip1 and both cycle withdrawal and differentiation are triggered after p27Kip1 level reaches a plateau. It has been postulated that this reflects a cycle counting mechanism mediated at a threshold level of p27Kip1 (Durand et al., 1997; Durand and Raff, 2000
). It was not resolved in these studies whether the role of p27Kip1 is only to trigger cycle arrest or is also directly involved in mechanisms leading to differentiation (Galderisi et al., 2003
). This distinction has been approached by experiments with CG-4 cells obtained from p27Kip1 null animals. Although CG-4 cells lack p27Kip1, a small fraction arrest and differentiate into normal astrocytes under deprived conditions of in vitro culture. These observations suggested that p27Kip1 primarily induces cell cycle arrest and only indirectly through cycle arrest does differentiation proceed (Tikoo et al., 1997
; Casaccia-Bonnefil et al., 1999
). In Xenopus, however, the homologous protein, p27Xic1, may be more directly required for specification of neural fate (Ohnuma et al., 1999
; Vernon and Philpott, 2003
). Whereas the protein operates as both an inhibitor of cycle progression and in cell fate specification, the active domains of the protein serving the two functions are only partially overlapping (Ohnuma et al., 1999
). These observations place p27Kip1, and perhaps related cdks, within a linkage that coordinates the mechanisms regulatory to specification and others regulatory to the proliferative process. The linkage of p27Kip1 overexpression, the associated advance in Q and modulation of the laminar fate are consistent with these observations from other experimental systems.
p27Kip1 and the Proliferative Model
Our proliferative model for neocortical neuronogenesis was formalized earlier (Caviness et al., 2000, 2003
; Nowakowski et al., 2002
) from observations based upon kinetic parameters and Q in normal embryos and subsequently confirmed and further validated in Ts16 trisomy (Haydar et al., 2000a
) and extended in the p27Kip1 overexpression model. l. This model (Figs 1A, 6AC) integrates the behavior of these parameters across the entire neuronogenetic interval in mouse. In brief, the founder population and progeny on average execute 11 integer cell cycles before exhaustion of proliferative activity in the PVE. The fractional advance of Q with each cell cycle determines the total number of cycles (11 in the normal mouse neocortex).
Pertinent to the present findings, the model predicts that should Q be increased at a rate ahead of its normal progression, there would be an early increase in cell cycle output (Caviness et al., 2003) (Fig. 6A,B). However, this would be at the expense of the size of the overall proliferative pool so that the total number of later formed neurons would be reduced (Fig. 6C). The effect should be more salient in down-gradient regions, i.e. more salient in less mature regions of the cerebral wall, than in up-gradient, more mature regions. These predictions are largely confirmed here where the rate of progression of Q with cell cycle has been increased by overexpression of p27Kip1 in the early part of the neuronogenetic interval. Thus, from the perspective of the size of the E14 labeled cohort counted in the P21 cortex, its size is increased in DT and to a greater degree in down-gradient field 1 than in upgradient field 40. However, in contrast to the size of this cohort directed to the cortex at E14, the total number of cells that will be directed to the cortex over the full remaining neuronogenetic interval is reduced (Table 3). The effect upon total cell numbers is detected only with respect to SG layers in field 1, which are largely being formed on and after E14. An effect is not detectable in field 40, which has largely completed its cycle of neuronogenesis by E14 (by which time a large proportion of cells has already been designated to SG layers).
Overall these studies illustrate the predominant importance of the parameter Q as determinant of the rate and number of neurons arising in the course of cell proliferation in the PVE. In principle, an accelerated progression of Q with successive cycles predicts an initial increased output per cycle, which then causes a reduction in the size of the proliferative population. This in turn leads to an ultimate reduction in total output (Caviness et al., 2003). In contrast, a retardation in the progression of Q with successive cycles predicts the opposite (Caviness et al., 2003
). Here Q was increased by only
10% at what was probably the point of maximum effect on E14 yet there was substantial reduction in the cells delivered to the cortex. In the p27Kip1/ (Fero et al., 1996
; Kiyokawa et al., 1996
; Nakayama et al., 1996
; Goto et al., 2004
) and the Ts16 mice (Haydar et al., 1996
, 2000a
) predictions of the opposite condition of retarded progression of Q with cycle are confirmed. In the p27Kip1/all other proliferative parameters, including founder population, cycle duration and growth fraction, are normal and the cortex is increased in cell number (Goto et al., 2005
). This consequence of the knockout indicates that p27Kip1 exerts a regulatory effect upon Q that is independent of that of other CDK inhibitors, at least in its progression through the earlier cycles of the neuronogenetic interval. Whereas our model scales Q to neuronal production, available observations relating to the coordinate interactions of CDK inhibitors do not yet allow a scaling of the independent contribution of p27Kip1 to the progression of Q.
In Ts16 the effect of changes in Q are offset by a smaller founder population and reduced growth fraction. The number of cells delivered through the early cycles is substantially reduced but the number largely normalizes with the later cycles (Haydar et al., 1996, 2000a
). Precocious acceleration rather than retardation, in the progression of Q with cell cycle in particular may underlie a wide range of genetically determined human disorders classified as microcephaly vera, as well as a host of other microcephalies that bear no indication of actively destructive pathological process (Mochida and Walsh, 2001
). In particular, relative attenuation of SG layers, as would be predicted from this model, is characteristic of certain forms of human microcephaly vera (Urich, 1976
).
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Beaulieu C (1993) Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res 609:284292.[CrossRef][ISI][Medline]
Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM (2004) p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 18:862876.
Boulder Committee (1970) Embryonic vertebrate nervous system: revised terminology. Anat Rec 166:257262.[CrossRef][ISI][Medline]
Cai L, Hayes NL, Takahashi T, Caviness VS Jr, Nowakowski RS (2002) Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior. J Neurosci Res 69:731744.[CrossRef][ISI][Medline]
Calegari F, Huttner WB (2003) An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. J Cell Sci 116:49474955.
Casaccia-Bonnefil P, Hardy RJ, Teng KK, Levine JM, Koff A, Chao MV (1999) Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation. Development 126:40274037.
Caviness VS (1975) Architectonic map of neocortex of the normal mouse. J Comp Neurol 164:247263.[CrossRef][ISI][Medline]
Caviness VS (1982) Neocortical histogenesis in normal and reeler mice: a developmental study based upon [3H]thymidine autoradiography. Dev Brain Res 4:293302.[ISI]
Caviness V, Takahashi T, Nowakowski R (1995) Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends Neurosci 18:379383.[CrossRef][ISI][Medline]
Caviness V, Takahashi T, Nowakowski R (2000) Neuronogenesis and the early events of neocortical histogenesis. In: Development of the neocortex (Goffinet A, Rakic P, eds), pp. 107143. Berlin: Springer Verlag.
Caviness VS, Goto T, Tarui T, Takahashi T, Bhide PG, Nowakowski RS (2003) Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process. Cereb Cortex 13:592598.
Delalle I, Takahashi T, Nowakowski RS, Tsai LH, Caviness VS Jr (1999) Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization. Cereb Cortex 9:824832.
Durand B, Raff M (2000) A cell-intrinsic timer that operates during oligodendrocyte development. Bioessays 22:6471.[CrossRef][ISI][Medline]
Durand B, Gao FB, Raff M (1997) Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation. EMBO J 16:306317.
Durand B, Fero ML, Roberts JM, Raff MC (1998) p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation. Curr Biol 8:431440.[CrossRef][ISI][Medline]
Fero M, Rivkin M, Tasch M, Porter P, Carow C, Firpo E, Polyak K, Tsai L-H, Broudy V, Perlmutter R, Kaushansky K, Roberts J (1996) A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell 85:733744.[CrossRef][ISI][Medline]
Galderisi U, Jori FP, Giordano A (2003) Cell cycle regulation and neural differentiation. Oncogene 22:52085219.[CrossRef][ISI][Medline]
Goto T, Mitsuhashi T, Takahashi T (2005) Altered patterns of neuron production in the p27Kip1 knockout mouse. Dev Neurosci (in press).
Grove EA, Fukuchi-Shimogori T (2003) Generating the cerebral cortical area map. Annu Rev Neurosci 26:355380.[CrossRef][ISI][Medline]
Hammond V, Tsai LH, Tan SS (2004) Control of cortical neuron migration and layering: cell and non cell-autonomous effects of p35. J Neurosci 24:576587.
Haubensak W, Attardo A, Denk W, Huttner WB (2004) Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc Natl Acad Sci USA 101:31963201.
Haydar TF, Blue ME, Molliver ME, Krueger BK, Yarowsky PJ (1996) Consequences of trisomy 16 for mouse brain development: corticogenesis in a model of Down syndrome. J Neurosci 16:61756182.
Haydar TF, Nowakowski RS, Yarowsky PJ, Krueger BK (2000a) Role of founder cell deficit and delayed neuronogenesis in microencephaly of the trisomy 16 mouse. J Neurosci 20:41564164.
Haydar TF, Wang F, Schwartz ML, Rakic P (2000b) Differential modulation of proliferation in the neocortical ventricular and subventricular zones. J Neurosci 20:57645774.
Hayes N, Nowakowski R (2000) Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering vs leaving the S-phase. Dev Neurosci 22:4455.[CrossRef][ISI][Medline]
His W (1904) Die Entwicklung des Menschlichen Gehirns wahrend der ersten Monate. Leipzig: von S. Hirzel.
Iacopetti P, Michelini M, Stuckmann I, Oback B, Aaku-Saraste E, Huttner WB (1999) Expression of the antiproliferative gene TIS21 at the onset of neurogenesis identifies single neuroepithelial cells that switch from proliferative to neuron-generating division. Proc Natl Acad Sci USA 96:46394644.
Kiyokawa H, Kineman R, Manova-Todorava K, Soares V, Hoffman E, Ono M, Khanam D, Hayday A, Frohman L, Koff A (1996) Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell 85:721732.[CrossRef][ISI][Medline]
Kosodo Y, Roper K, Haubensak W, Marzesco AM, Corbeil D, Huttner WB (2004) Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J 23:23142324.
Livesey FJ, Cepko CL (2001) Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neurosci 2:109118.[CrossRef][ISI][Medline]
Mallamaci A, Muzio L, Chan CH, Parnavelas J, Boncinelli E (2000) Area identity shifts in the early cerebral cortex of Emx2/ mutant mice. Nat Neurosci 3:679686.[CrossRef][ISI][Medline]
McConnell SK (1989) The determination of neuronal fate in the cerebral cortex. Trends Neurosci 12:342349.[CrossRef][ISI][Medline]
Mitsuhashi T, Aoki Y, Eksioglu YZ, Takahashi T, Bhide PG, Reeves SA, Caviness VS, Jr. (2001) Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells. Proc Natl Acad Sci USA 98:64356440.
Miyama S, Takahashi T, Nowakowski RS, Caviness V (1997) A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium. Cereb Cortex 7:678689.[Abstract]
Mochida GH, Walsh CA (2001) Molecular genetics of human microcephaly. Curr Opin Neurol 14:151156.[CrossRef][ISI][Medline]
Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh D, Nakayama K-I (1996) Mice lacking p27Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85:707720.[CrossRef][ISI][Medline]
Nowakowski R, Lewin S, Miller M (1989) Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population. J Neurocytol 18:311318.[CrossRef][ISI][Medline]
Nowakowski R, Caviness V Jr, Takahash IT, Hayes N (2002) Population dynamics during cell proliferation and neuronogenesis in the developing murine neocortex. In: Cortical development: from specification to differentiation (results and problems in cell differentiation) (Hohmann C, ed.), pp. 122. New York: Springer-Verlag.
O'Leary DD, Nakagawa Y (2002) Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex. Curr Opin Neurobiol 12:1425.[CrossRef][ISI][Medline]
Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA (1999) p27Xic1, a Cdk inhibitor, promotes the determination of glial cells in Xenopus retina. Cell 99:499510.[CrossRef][ISI][Medline]
Ohnuma S, Philpott A, Harris WA (2001) Cell cycle and cell fate in the nervous system. Curr Opin Neurobiol 11:6673.[CrossRef][ISI][Medline]
Ohnuma S, Hopper S, Wang KC, Philpott A, Harris WA (2002) Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina. Development 129:24352446.[ISI][Medline]
Ohshima T, Ogawa M, Veeranna, Hirasawa M, Longenecker G, Ishiguro K, Pant HC, Brady RO, Kulkarni AB, Mikoshiba K (2001) Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain. Proc Natl Acad Sci USA 98:27642769.
Rakic P (1976) Differences in the time of origin and in eventual distribution of neurons in areas 17 and 18 of visual cortex in Rhesus monkey. Exp Brain Res Suppl 1:244248.
Ren JQ, Aika Y, Heizmann CW, Kosaka T (1992) Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons. Exp Brain Res 92:114.[ISI][Medline]
Sauer FC (1935) Mitosis in the neural tube. J Comp Neurol 62:377405.[CrossRef]
Sauer FC (1936) The interkinetic migration of embryonic epithelial nuclei. J Morphol 60:111.[CrossRef]
Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:15011512.
Shin DM, Korada S, Raballo R, Shashikant CS, Simeone A, Taylor JR, Vaccarino F (2004) Loss of glutamatergic pyramidal neurons in frontal and temporal cortex resulting from attenuation of FGFR1 signaling is associated with spontaneous hyperactivity in mice. J Neurosci 24:22472258.
Sidman RL, Rakic P (1973) Neuronal migration, with special reference to developing human brain: a review. Brain res 62:135.[CrossRef][ISI][Medline]
Takahashi T, Nowakowski RS, Caviness VS Jr (1994) Mode of cell proliferation in the developing mouse neocortex. Proc Natl Acad Sci USA 91:375379.
Takahashi T, Nowakowski R, Caviness V (1995) The cell cycle of the pseudostratified ventricular epithelium of the murine cerebral wall. J Neurosci 15:60466057.[Abstract]
Takahashi T, Nowakowski R, Caviness V (1996a) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general computational model of neocortical neuronogenesis. J Neurosci 16:61836196.
Takahashi T, Nowakowski RS, Caviness VS Jr (1996b) Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. J Neurosci 16:57625776.
Takahashi T, Goto T, Miyama S, Nowakowski RS, Caviness VS Jr (1999) Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall. J Neurosci 19:1035710371.
Takahashi T, Nowakowski R, Caviness V (2000) Neocortical neuronogenesis: regulation, control points and a strategy of structural variation. In: The handbook of developmental cognitive neuroscience (Nelson C, Luciana M, eds), pp. 323. Cambridge, MA: MIT Press.
Theiler K (1972) The house mouse. Development and normal stages from fertilization to 4 weeks of age. Berlin: Springer Verlag.
Tikoo R, Casaccia-Bonnefil P, Chao M, Koff A (1997) Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells. J Biol Chem 272:442447.
Urich H (1976) Malformations of the nervous system, perinatal damage and related conditions in early life. In: Greenfield's neuropathology (Blackwood W, Corsellis J, eds), pp. 361469. London: Edward Arnold.
Verney C, Takahashi T, Bhide P, Nowakowski RS, Caviness V (2000) Independent controls for neocortical neuron production and histogenetic cell death. Dev Neurosci 22:125138.[CrossRef][ISI][Medline]
Vernon AE, Philpott A (2003) A single cdk inhibitor, p27Xic1, functions beyond cell cycle regulation to promote muscle differentiation in Xenopus. Development 130:7183.
Vernon AE, Devine C, Philpott A (2003) The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus. Development 130:8592.
Walsh CA (2000) Genetics of neuronal migration in the cerebral cortex. Ment Retard Dev Disabil Res Rev 6:3440.[CrossRef][ISI][Medline]
Watanabe G, Pena P, Shambaugh GE 3rd, Haines GK 3rd, Pestell RG (1998) Regulation of cyclin dependent kinase inhibitor proteins during neonatal cerebella development. Brain Res Dev Brain Res 108:7787.[ISI][Medline]
Zindy F, Cunningham JJ, Sherr CJ, Jogal S, Smeyne RJ, Roussel MF (1999) Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases. Proc Natl Acad Sci USA 96:1346213467.