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

I. Delalle1, T. Takahashi1,2, R.S. Nowakowski3, L.-H. Tsai4 and V.S. Caviness, Jr1

1 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA , , 2 Department of Pediatrics, Keio University School of Medicine, Tokyo 160, Japan, , 3 Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 and , 4 Department of Pathology and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, 02114, USA

Address correspondence to Ivana Delalle, Department of Neuropathology, Massachusetts General Hospital, WRN3 Fruit Street, Boston, MA 02114, USA. Email: idellale{at}partners.org.


    Abstract
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.


    Introduction
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurons of the neocortex arise from a proliferative pseudostratified epithelium (PVE) (Takahashi et al., 1995aGo) which lines the supraventricular surface of the embryonic cerebral wall. The proliferative process is regulated coordinately with tight precision across the epithelium. Thus, the onset, progression and termination of neuronogenesis advance systematically from rostrolaterally to caudomedially across the vast expanse of the PVE (Smart and McSherry, 1982; Smart and Smart, 1982) according to a ‘transverse neurogenetic gradient' (Bayer and Altman, 1991Go). In mouse, independently of position within the transverse neurogenetic gradient, neuronogenesis occurs during a 6 day period (the neuronogenetic interval or NI). During this interval the founder population and its progeny in any position in the PVE execute 11 cell cycles (Takahashi et al., 1995aGo; Miyama et al., 1997Go). With each successive cycle, there is an increase in the duration of the cell cycle (TC), due entirely to an increase in the duration of the G1 phase of the cycle (TG1), and an increase in the proportion of postmitotic cells that exits each cycle, i.e. the fraction Q. These observations direct attention to the molecular regulation of Q and TG1 as the control point(s) for the overall neuronogenetic process (Caviness et al., 1999Go), and available evidence [reviewed by Caviness and co-workers (Caviness et al., 1995Go, 1999Go)] suggest this to be the case in other mammals including monkey (Kornack and Rakic, 1992Go, 1998Go). In monkey, for example, there are ~28 cell cycles in the NI (rather than 11 in the mouse), resulting in a vast increase in the multiplier power of the proliferative process in this species in comparison to mouse (Caviness et al., 1995Go, 1999Go).

We present here a quantitative analysis of expression patterns of mRNAs of a set of five proteins known to be critical to regulation of the vertebrate cell cycle. These are the kinase cdk2 and its regulatory subunit cyclin E, both essential to the G1 to S transition (Knoblich and Lehner, 1993Go; Koff et al., 1992Go; Sherr, 1996Go; Tsai et al., 1993aGo); the cycle inhibitory proteins p27 and p21, whose actions are directed at the G1 restriction point (Hunter, 1993Go; Xiong et al., 1993Go; Polyak et al., 1994Go); and cyclin B, which is known to act at the G2 to M phase transition (Sherr and Roberts, 1995Go). The proteins chosen for analysis represent only a limited sampling of the proteins known to be involved in regulation of the vertebrate cell cycle. The choice of cdk2 and cyclin E and B is influenced by the general belief that these proteins are known to be indispensable to vertebrate cycle progression (Koff et al., 1992Go; Knoblich and Lehner, 1993Go; Sherr, 1996Go; Tsai et al., 1993aGo). In addition, our choices were limited to proteins known to be present and active in the proliferative cells of the murine neocortical PVE (Tsai et al., 1993bGo; Fero et al., 1996Go; Kiyokawa et al., 1996Go; Lee et al., 1996Go; Nakayama et al., 1996Go). The immediate objective of the analysis is to correlate patterns of expression of the mRNAs of these proteins with the dynamics of change of the proliferative process. Such correlations may be expected to guide future investigative strategies which will seek to identify the molecular mechanisms of neuronogenetic regulation.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animals

Brains of CD1 mice were obtained on embryonic days (E) 12, 13 and 15 (E0 = day of conception). The brains, fixed in 4% neutral buffered paraformaldehyde, were prepared for sectioning by cryostat at 12 µm intervals (Delalle et al., 1997Go).

Riboprobes

Total RNA from E10 mouse embryos was isolated by guanidinium thiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987Go). First strand cDNA was obtained by reverse transcription upon incubation of the mouse RNA with primer oligo (dT) 15. In order to make the most specific riboprobes for mRNAs of interest, sequences of primers were chosen so as to flank the most nearly unique and specific parts (~220–350 bp) of the cDNA region encoding each of the respective cell cycle proteins (cyclin E, X75888: 927–1227; cdk2, U63337: 849–1016; cyclin B, X58708: 234–530). 3' and 5' primers (length ~30 bp) were synthesized so as to include EcoRI and BamHI restriction sites respectively on their 5' ends. Upon digestion with the appropriate restriction enzymes, these most specific parts of cDNA for each of the proteins above were cloned into pBSK+/– phagemid (Strategene) vectors from which [35S]UTP-labeled antisense and sense riboprobes were synthesized using T7 and T3 RNA polymerases respectively. DNA templates obtained by polymerase chain reaction for cyclin E, cdk2 and cyclin B riboprobes were sequenced and found to show 99% identity with chosen, unique parts of respective cDNAs and yielded specific alignments in the BLAST search.

For p27 Kip1 (p27) antisense and sense probes were synthesized from pcDNA3 vector (Invitrogen) containing 700 bp of mouse p27 cDNA fragment (provided by Dr L.-H. Tsai) using Sp6 and T7 RNA polymerases respectively. For p21Cip1 (p21), antisense and sense probes were synthesized with T7 RNA polymerase from pT7Blue® vectors (Novagen) containing specific segments (350 bp) of mouse p21 cDNA in opposite orientations (gift of Dr P. Dotto).

In Situ Hybridization

Optimum hybridization conditions were established empirically for each of the riboprobes through a series of trials with several specimens from embryos of each age. The optimized conditions at each age were then applied in a standard way and all hybridization procedures for each riboprobe were run concurrently so that measures of grain density for each probe could be compared quantitatively from animal to animal at the same age and across embryonic ages.

Prehybridization

Tissue sections were fixed in buffered 4% paraformaldehyde (5 min), rinsed twice in PBS (5 min each), immersed in 0.1 M triethanolamine– HCl (10 min) and then in 0.25% acetic anhydride in 0.1 M triethanolamine–HCl (10 min), rinsed in 2 x SSC (5 min), dehydrated through 70, 80, 95 and 100% ethanol (1 min each), immersed in chloroform (twice, 5 min each), rinsed in 100 and 95% ethanol (1 min each) and air-dried.

Hybridization

Hybridization buffer contained 50% deionized formamide, 0.3 M NaCl, 20 mM Tris, 5 mM EDTA, 10 mM NaH2OPO4–H2O, pH 8.0, 1 x Denhardt's solution, 10% dextran sulfate, 0.5 mg/ml yeast tRNA and 10 mM DTT. The probe concentration was always 50 000 c.p.m./ml. Between 20 and 50 ml of the hybridization solution was applied per section depending on the size of section and covered with coverglass. Slides were incubated in a humid chamber for 18 h at 50–53°C.

Washing Procedure

Following rinsing in 2 x SSC four times (1 min each), sections were incubated for 30 min in RNAseA solution (20 mg/ml in RNAse buffer) at 37°C and washed afterwards for 30 min in RNAse buffer (29.22 g NaCl; 10 ml 1 M Tris–HCl, pH 8.0; 2 ml 0.5M EDTA, pH 8.0, in 1 l of deionized water) at room temperature. Sections were then washed in decreasing concentrations of SSC (2x, 0.5x and 0.2x) for 30 min periods at 55°C, dehydrated in increasing concentrations of ethanol (50, 75, 95 and 100%) and air-dried.

Autoradiography

All slides for a given probe for the full series of brains at the three embryonic ages were processed together through all autoradiographic procedures so as to minimize variations in quantitative grain count recovery that might be associated with differences in autoradiographic exposure conditions. They were coated with Kodak NTB2 liquid emulsion, diluted 1:1 with deionized distilled water. After 2 weeks at 4°C slides were developed and counterstained in 0.1% basic fuchsin solution.

Quantitative Signal Analysis

All experimental and histological procedures for each probe were executed together so as to be subject to a common set of experimental sources of variation in autoradiographic signal. This approach allows estimates of the relative variation in signal for each probe among experimental animals. Obviously, signal intensity of a given mRNA cannot be related to the amount of corresponding protein that will be synthesized or its activity (Zhang et al., 1994Go; Pagano et al., 1995Go; LaBaer et al., 1996Go; Vlach et al., 1997Go). Nor can the relative signal intensities across the diverse probes be taken as an indicator of the relative amounts of protein or their activities among the diverse set of proteins (Delalle et al., 1997Go).

Despite these limitations, other considerations argue that these measures of mRNA signal intensity probably provide a meaningful estimate across time of the relative, though not absolute, abundance and activity of the corresponding protein in cells of the PVE. In part this reflects favorable properties of the PVE itself. Firstly, all cells of the PVE not only are proliferative [i.e. the growth fraction is essentially 1.0 throughout the neuronogenetic interval (Takahashi et al., 1995aGo; Miyama et al., 1997Go)], but the kinetic parameters among cells of the population at each region are minimally variant (Cai et al., 1997Go). Thus, values obtained in a given region at a give time represent those for a uniformly proliferative cell population. Secondly, the density of cell nuclei across the width of the PVE varies <10% and is uniform in this way throughout the neuronogenetic interval across the regions to studied here (Miyama et al., 1997Go; Takahashi et al., 1992Go, 1993Go, 1995aGo, Takahashi et al., bGo). Thus, values obtained in each region sampled and across embryonic time are referable to populations of equivalent density. The proteins chosen are also favorable for such quantitative treatment. This is particularly the case for the cyclins E and B. For these cyclins both abundance and activity have been found to be regulated by transcription and degradation on a cycle-by-cycle basis in all proliferative vertebrate cell systems (Koff et al., 1992Go; Knoblich and Lehner, 1993Go; Morgan, 1995Go; Pagano et al., 1995Go; Sherr and Roberts, 1995Go; Hengst and Reed, 1996Go). Thus, both are fully synthesized and destroyed within limited segments of a single cell cycle with no carry-over into successive cycles. For the others, transcription has been a meaningful index of protein synthesis but less so for abundance and activity. In particular the non-cyclin proteins considered here have half-lives that are probably longer than the single cell cycle and so will accumulate over the course of multiple cycles (Zhang et al., 1994Go; Pagano et al., 1995Go; LaBaer et al., 1996Go; Vlach et al., 1997Go). Their activities probably reflect their abundance. For these reasons we provisionally accept signal intensity variations for the mRNAs of each of these proteins to represent meaningful indices of relative abundance and activity of that protein across the NI among the respective sets of animals. It is probable that correspondence between transcription and protein abundance and activity is closest for the cyclins (Koff et al., 1992Go; Knoblich and Lehner, 1993Go; Sherr and Roberts, 1995Go). For the other proteins mRNA signal intensity very likely underestimates protein abundance and activity, and this disparity probably advances with successive cell cycles. We caution, moreover, that the relative signal intensities for the mRNAs on a given protein carry no implications for the abundance of this protein in comparison to the other proteins, even when comparisons are made at the same age and in the same region of the PVE (Delalle et al., 1997Go).

The sections were examined under a x40 objective lens in a dark-field light microscope fitted with a high-performance color CCD camera (Sony). Autoradiographic grain densities were determined in digitized microscopic images using IPLab Spectrum (image analysis software for the Macintosh). For each section analyzed, grain counts were made across the full width of a standard sector of the cerebral wall (Miyama et al., 1997Go) where the width of the sector was 72 µm in its medial to lateral dimension and 12 µm in thickness corresponding to section thickness. The radial dimension of the cerebral wall (ventrodorsal axis, perpendicular to the ventricular surface) was subdivided into bins 11 µm in height numbered 1, 2, 3, etc. from the ventricular surface outward and with each bin parallel to the ventricular surface (Takahashi et al., 1992Go). Counts were made in this way for each probe on 3–5 pairs of sections (one section in a pair hybridized with the antisense and the other with the sense probe) coming from at least two different brains and from at least two different litters. Counts assigned to mRNAs correspond to counts with antisense probes – (counts with sense probes + background) where all counts are made on equal specimen areas. Thus, counts could be expressed as autoradiographic grain densities for an entire standard sector of the cerebral wall or as densities within single or sets of bins from the standard sectors.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The analyses presented here were conducted in lateral (LCZ) and dorsomedial (DCZ) zones of coronal sections of the developing cerebral wall from brains of embryos fixed for histological procedures at approximately 09.00 h on each of E12, E13 and E15 (Takahashi et al., 1995aGo; Miyama et al., 1997Go) (Fig. 1Go, Table 1Go). The radial span of the cerebral wall includes the ventricular zone (VZ) where effectively 100% of the cells are proliferative (Takahashi et al., 1995aGo); the intermediate zone (IZ), which includes a mix of migrating neurons and secondary proliferative populations; and the cortex, which is a concentration of postmigratory cells in initial stages of growth and differentiation (Takahashi et al., 1995bGo, 1996aGo,Takahashi et al., cGo; Miyama et al., 1997Go). The embryonic age range E12–E15 samples the patterns and levels of expression of these mRNAs through almost seven cell cycles of the 11 cell cycle murine NI. This is nearly 70% of the integer cell cycles of the NI and includes >70% of the full range of values of Q and TG1 occurring over the full neuronogenetic interval (Table 1Go). Importantly, this range of Q and TG1 includes cell cycles 3–9, which in mouse are the cycles in which the cycle-to-cycle increments in Q and TG1 are greatest (Takahashi et al., 1995aGo, 1996bGo). Because DCZ and LCZ are widely separated along the transverse neurogenetic gradient (Miyama et al., 1997Go), there is the critical analytical advantage that within a single histological section at each of the ages E12–E15 the experiment samples patterns and levels of mRNA expression representative of substantially different stages of advance of the proliferative process (Table 1Go).



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Figure 1.  The neuronogenetic gradient of the mouse neocortex. (A) Neuronogenesis in the neocortical pseudostratified ventricular epithelium (PVE) is initiated rostrolaterally and advances caudomedially as development progresses. (B) The present analyses were undertaken approximately at midhemisphere in lateral (LCZ) and dorsomedial (DCZ) regions of the embryonic murine neocortical cerebral wall (Miyama et al., 1997Go).

 

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Table 1
 
Distributions with Respect to Strata of Cerebral Wall

Signal for the mRNA for each protein is detected in all strata of the DCZ and LCZ throughout the interval E12–E15 (Fig. 2Go: cyclin E and p27 are illustrated). There were no obvious differences in the stratification of the labeling patterns observed either at different ages or between the LCZ and DCZ of the same specimen. A preponderance of the total signal for the mRNAs for cyclins B and E and for cdk2 is detected in the VZ. This bias to the VZ is particularly prominent for the LCZ at E12 and for LCZ and DCZ at E13 (Fig. 3Go). The predominance of mRNA of these cell cycle related proteins in the proliferative VZ is in accord with previously demonstrated patterns of distribution of the corresponding proteins and of their histone H1 kinase activities in proliferating cells including analyses of the murine neocortical PVE (Tsai et al., 1993bGo; Lee et al., 1996Go).



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Figure 2.  Micrographs of autoradiograms of in situ hybridization of cyclin E and p27 mRNA. Cyclin E: sense control at E12 (A); antisense probe at low magnification of full coronal view at E12 (B); antisense probe at higher magnification of DCZ (C) and of LCZ (D) at E12; antisense probe at higher magnification of LCZ at E15 (E). In this cyclin E series the strongest signal is in the VZ with reference to background signal level in the sense control. Signal in the VZ is much stronger in the E12 than the E15 preparation, and at E12 it is stronger in DCZ than LCZ. p27: E12 sense control (F), E12 antisense of DCZ (G) and at E13 of LCZ (H). Signal intensity at E12 in DCZ is above background in the sense control but is much higher in LCZ at E13. For the cyclin E and p27 series, dashed lines parallel to the cerebral surface in (A), (C), (D), (F), (G) and (H) mark the division between the ventricular zone (vz) and primitive plexiform zone (ppz) at the outer surface of the cerebral wall. Dashed lines parallel to the cerebral surface in (E) subdivide the cerebral wall into vz, subventricular zone (svz), intermediate zone (iz), cortical plate (cp) and marginal zone (mz). Dashed lines perpendicular to the cerebral surface in these micrographs indicate the location of analyzed sectors of the cerebral wall. Darkfield illumination. Bar = 250 µm in (B) and 62.5 µm elsewhere.

 


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Figure 3.  Proportionate mRNA signal in the neocortical VZ. The counts for the mRNA for cyclins B and E, cdk2, p27 and p21 in the VZ are expressed as a percentage of total counts. These percentages determined in both DCZ (D) and LCZ (L) are plotted as histograms for E12, E13 and E15.

 
Low levels of signal for the mRNA for cyclin E and cdk2 at E15 but higher levels for p21 and p27 at E12-E15 are also detected in the subventricular zone (SVZ)-IZ and in the cortical strata. The SVZ-IZ contains proliferative cells. Both SVZ-IZ and cortical zone also include non-proliferating cells, some of which are migrating and some of which are postmigratory and differentiating. For p21 and p27 the proportionate mRNA signal is essentially equally distributed to VZ, on the one hand, and the SVZ-IZ and cortical strata, on the other, at E15 (Fig. 3Go). Thus, transcripts of these proteins may be formed in non-proliferating cells in both the SVZ-IZ and cortical strata. Transcription of these cell cycle related proteins in strata containing non-proliferative cells has been observed by other investigators (Heintz, 1993Go; Tsai et al., 1993aGo; Zhang et al., 1994Go; Halevy et al., 1995Go; Parker et al., 1995Go; Lee et al., 1996Go).

Quantitative Analysis of Signal within the VZ

Expression of mRNA in Relation to Cell Cycle Phases

The principal proliferative population of the VZ corresponds to the pseudostratified ventricular epithelium. Essentially all of the cells in this population are proliferating and also the proliferating cells in this population are asynchronously distributed such that the proportion of cells in a given phase of the cycle corresponds to the duration of that phase relative to the duration of the complete cell cycle. Thus, at E12 cells in G1, S and G2 + M phases are approximately 35, 45 and 20%; on E13, approximately 55, 30 and 15%; and on E15, approximately 60, 30 and 10% respectively (Takahashi et al., 1995aGo; Miyama et al., 1997Go). The nuclei of cells of this epithelium move radially upward and downward across the width of epithelium in the course of the cell cycle (Sauer, 1935Go, 1936Go; Sidman et al., 1959Go; Takahashi et al., 1995aGo). Thus, the somata of cells in M phase are concentrated at the ventricular margin, while S phase occurs with the nucleus located in the outer half of the epithelium. Nuclei of cells in G1 phase are distributed throughout the full width of the epithelium, while nuclei of cells in G2 phase are found largely concentrated in the middle third of the epithelium, overlapping those of cells in S phase in the outer half but also intermixed with those in M phase at the ventricular margin (Takahashi et al., 1995aGo). A proportion of cells within the VZ, corresponding to the cells of the fraction Q, will also be postproliferative young neurons which are exiting the cycle. Although Q increases rapidly from E12 to E15, this population is at all times small relative to the total population of the VZ as reflected in the observations that the growth fraction is essentially 1.0 throughout (Waechter and Jaensch, 1972Go; Takahashi et al., 1995aGo; Miyama et al., 1997Go) and the fact that the Q cells leave the VZ within a few hours of their final mitosis (Takahashi et al., 1996aGo).

The mRNAs of cdk2, p21 and p27 are distributed more or less uniformly across the full width of the VZ on each of E12–E15 (Fig. 4Go). This implies that these three proteins are transcribed through all phases of the cell cycle. The mRNAs for cyclins E and B, by contrast, are differentially expressed with respect to depth in the VZ (Figs 2 and 4GoGo). Cyclin E is somewhat more abundant in the inner than in the outer half of the VZ, which is consistent with its expression as the proliferative cells approach the G1/S transition (Koff et al., 1992Go; Takahashi et al., 1992Go, 1995aGo; Tsai et al., 1993aGo; Sherr and Roberts, 1995Go) (Fig. 4Go). The expression of mRNA for cyclin B, by contrast, is weighted somewhat to the outer half of the VZ, where a majority of cells are in S phase and G2 phase (Takahashi et al., 1992Go, 1995aGo). This pattern is consistent with expression in cells in transit from S into M phase and principally in G2 phase (Lew et al., 1991Go; Knoblich and Lehner, 1993Go; Sherr and Roberts, 1995Go). These patterns of expression of mRNAs of the five proteins with respect to cell cycle phases in the VZ are consistent with generally established patterns of regulation of cdk2, cyclin E, cyclin B, p21 and p27 synthesis and activity in vertebrate cells (Lew et al., 1991Go; Koff et al., 1992Go; Knoblich and Lehner, 1993Go; Tsai et al., 1993aGo; Sherr, 1994Go; Zhang et al., 1994Go; Sherr and Roberts, 1995Go; LaBaer et al., 1996Go; Vlach et al., 1997Go). This consistency validates the quantitative in situ methods as applied here to the embryonic murine cerebral wall.



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Figure 4.  Distribution of mRNA signal on E12 in the DCZ of the neocortical VZ. The counts for the mRNA for cyclins B and E, cdk2, p27 and p21 are represented as a function of bin location in the DCZ of the neocortical VZ. The full width of the VZ is divided into 12 bins 11 µm in height and numbered 1, 2, 3, etc. from the ventricular surface outward. Similar distributions are seen in LCZ and the distributions seen in DCZ and LCZ are similar on each embryonic day.

 
It should be realized that because in the outer part of the PVE cells in G1 are abundantly intermixed with cells in S, and in the inner part of the PVE the cells in G2 are intermixed with cells in G1 and M phases, the magnitude of the measured mRNA surge for cyclins E and B in outer and inner regions of the PVE, respectively, undoubtedly underestimates greatly the actual surges of mRNA expression in these phases of the cell cycle. Moreover, because of the substantial increase in the proportion of cells in G1 phase over the E12–E15 interval, the proportionate drop in level of expression of cyclin E mRNA is greatly under-estimated with respect to the proportion of cells in G1 phase. These shifts in cell proportions with respect to cycle phase are not significant for p27 expression because this continues through all phases of the cell cycle. As noted above, at all times the proportion Q of the population within the VZ is low and, thus, the expression of these mRNAs in postproliferative cells will not significantly alter the apparent expression in the proliferative population.

Total Signal in Relation to Embryonic Date : Two Patterns of mRNA Expression

The total grain counts recovered from the VZ for the mRNAs of cyclin E, cyclin B and p27 vary several-fold during the developmental period analyzed here; the levels of expression of the mRNAs for cdk2 and p21, by contrast, vary only some 30% during this period (Table 1Go). Variation in mRNA expression over the E12–E15 interval follows two general patterns, one where expression is maximum at the outset of neuronogenesis and falls subsequently (cyclin E and p21) and a second where expression is minimum at the outset of neuronogenesis, increase later and then declines again (p27, cdk2 and cyclin B) (Fig. 5Go).



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Figure 5.  Expression patterns of mRNAs across the neuronogenetic interval. Mean counts (bars = SEM) within the VZ for mRNA of cyclin E and p27 (A) and of cdk2, p21 and cyclin B (B) are expressed as functions of both cell cycle number and Q. The interval surveyed in these experiments includes the greater part of the full progression of Q from 0.0 to 1.0 in the mouse neocortical PVE. The data points are a synthesis from counts made in both the dorsal (DCZ) and lateral (LCZ) regions of the cerebral wall (Table 1Go). On each of the embryonic days E12, E13 and E15 the values of Q and the integer cell cycle in the LCZ will be higher than these parameters in the DCZ.

 
    Pattern 1. Grain count densities for cyclin E and p21 mRNAs over the VZ are at their maxima on E12 in DCZ (cycle 2.7). Grain count densities of cyclin E mRNA subsequently fall ~70% within the same sections to a sustained low level with the level measured at LCZ on E12 (cycle 5.1), approaching a low asymptote. Grain count densities of cyclin E mRNA change relatively little with progression of the NI through E13 and E15 (through cycle 9.8). Grain count densities for p21 mRNA fall only ~30% overall, with the rate of fall more or less uniform over the E12–E15 interval.

    Pattern 2. Grain count densities for p27, cdk2 and cyclin B mRNA over the VZ are at their minima on E12 in DCZ but are somewhat higher on E12 in LCZ. The p27 densities then surge on E13 within the same sections with cycle transition from DCZ (still at cycle 5.0) to peak densities attained for the overall NI in LCZ (cycle 6.9). For the mRNAs of p27 this corresponds to an increase of ~300% relative to the levels on the LCZ on E12 with the major differential in the comparison of values in DCZ and LCZ on E13. Grain count densities for this set of mRNAs are substantially lower on E15 with relatively little difference in grain count densities in DCZ and LCZ. The patterns for grain count densities of cdk2 and cyclin B mRNA follow those of p27; the amplitude variations for mRNA of cyclin B are comparable to those for p27 but less variable for that of cdk2.

Importantly, these variations in pattern of expression advance systematically as a function of cell cycle, i.e. of the state of progression of the proliferative process in the PVE. They correlate less well with embryonic time (Table 1Go). In fact, with the mRNAs of both of these patterns the most extreme differences in grain density are encountered in comparison to values in LCZ and DCZ at the same developmental age (for cyclin E and p21 between DCZ and LCZ at E12; for p27, cdk2 and cyclin B between DCZ and LCZ at E13), i.e. within the same coronal sections. The finding that cyclin E expression is maximum at the outset of neuronogenesis but subsequently falls sharply is consistent with our prior observation that cdk2 histone H1 kinase activity (which is dependent upon cyclin E expression) follows this same pattern (Tsai et al., 1993bGo). The finding that p27 expression advances from barely detectable levels early in neuronogenesis to high levels at the end of neuronogenesis has been previously established by Western blot analysis (Lee et al., 1996Go). Moreover, the patterns of expression of these proteins are entirely consistent with what would be predicted from the previously documented differences in the developmental state of the DCZ and the LCZ at E13 (Miyama et al., 1997Go). This concordance between predicted and observed findings also means that variable conditions of histological and in particular autoradiographic processing cannot account for the variations in grain count density because the different parts of the same section are clearly processed identically.


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two Patterns of mRNA Expression: Three Phases of Neuronogenesis

Each of the cell cycle related proteins considered here has previously been demonstrated to be present and active in the rodent neocortical PVE (Tsai et al., 1993bGo; Fero et al., 1996Go; Kiyokawa et al., 1996Go; Lee et al., 1996Go; Nakayama et al., 1996Go). Here we distinguish two patterns of variation over the course of the NI in expression of the corresponding mRNAs (Figs 5 and 6GoGo). Provisionally we take these patterns of mRNA expression to represent, at the minimum, indices of the relative abundance of the corresponding protein as these vary across the NI. Thus, it has been established that generally in proliferative vertebrate cells the presence of cyclins E and B are regulated principally by transcription and degradation by being spread around on a cycle-by-cycle basis such that these proteins do not accumulate from cycle to cycle (Morgan, 1995Go; Pagano et al., 1995Go; Hengst and Reed, 1996Go). The proteins p27, p21 and cdk2, by contrast, are not so discretely formed and eliminated in the course of single cell cycles and may accumulate in proliferative cells over the course of successive cycles (Morgan, 1995Go; Pagano et al., 1995Go; Hengst and Reed, 1996Go).



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Figure 6.  Three phases of cyclin E and p27 mRNA expression across the neuronogenetic interval and their relation to the proliferative parameters Q and the duration of the G1 phase (TG1) as described previously (Miyama et al., 1997Go). The amplitude of each parameter is normalized on the ordinate as a percentile where maximum values for each are 100%. Progression of the neuronogenetic interval is expressed on the abscissa as elapsed integer cycles of the total series of 11 cycles in mouse. In this figure we represent only the patterns of cyclin E and p27 mRNA expression. These patterns are extended by extrapolation from best fit curves over the full 11 cycles of the NI partitioned with respect to the three phases of mRNA expression. The plots are limited to patterns of mRNA expression of cyclin E and p27, but they are intended to be representative of other mRNAs, including cdk2 and p21, but also with a larger sets of proteins which regulate the progression of the vertebrate cell cycle and in particular the progression of the G1 phase of the cycle.

 
The two patterns of mRNA expression partition the NI into three phases as represented in Figures 5 and 6GoGo where the progression of the NI may be conceived in terms of cell cycle succession rather than in terms of embryonic age. Previous work has established that the two variable proliferative parameters, Q and TG1, vary systematically with cell cycle rather than with embryonic age (Miyama et al., 1997Go; Takahashi et al., 1999Go). Figures 5 and 6GoGo and Table 1Go demonstrate that mRNA expression also varies systematically with cell cycle, and the systematic variation defines three phases which can be correlated with previously demonstrated variations in Q and TG1 as follows:

Phase I (Cell Cycles 1–5)

Cyclin E mRNA expression is initially high but falling, while p27 mRNA expression is initially low but rising; over the interval investigated here Q and TG1 are initially at their minima, 0.09 and 3.3 h, and increase only to 0.25 and 5.5 h respectively.

Phase II (Cell Cycles 6–8)

Cyclin E mRNA expression remains at low asymptote but that of p27 reaches and remains at a high level; Q increases rapidly to 0.5 and beyond, while TG1 increases even more rapidly to approach high asymptote at 8.8 h.

Phase III (Cell Cycles 9–11)

The expression of cyclin E mRNA remains low and that of p27 falls again; Q increases rapidly to 1.0. TG1, already at high asymptote by cycle 9, reaches its maximum duration.

These three phases are defined and will be discussed with respect to patterns of mRNA expression of cyclin E and p27 in part because the amplitudes of variation in patterns of expression of these mRNAs are the most dramatic. A more substantial reason, however, is that both of these proteins have previously been established to be at least partially rate-limiting operators in critical transitions relating to regulation of the G1 phase of the vertebrate cell cycle (Tsai et al., 1993aGo; Sherr and Roberts, 1995Go; Sherr, 1996Go). In considering the cell biological events occurring during these three phases, it should be kept in mind, however, that cyclin E and p27 not only interact directly or indirectly with cdk2 and p21 but also with a larger sets of proteins which regulate the progression of the vertebrate cell cycle and in particular the progression of the G1 phase of the cycle (Koff et al., 1992Go; Sherr, 1994Go; Massague and Polyak, 1995Go; LaBaer et al., 1996Go; Sheaff et al., 1997Go).

Phasic Progression of Neuronogenesis and the G1 Phase of the Cell Cycle

The parameters Q and TG1, which vary as illustrated in Figures 5 and 6GoGo with the phasic progression of cyclin E and p27 mRNA expression, govern the progression of the proliferative process of the murine neocortical PVE. Q corresponds to the probability that a postmitotic cell will exit the cycle at the G1 restriction point. The pattern of increase of Q with successive cell cycles governs the multiplier power of the founder population. It sets the rate of cell output per cycle and the overall cumulative output from the full succession of cycles from this founder population (Takahashi et al., 1996bGo).

TG1 is the only cell cycle phase duration which varies over the NI of the murine neocortical PVE (Takahashi et al., 1995aGo). Advance in TG1 exclusively governs the advance of overall cell cycle duration which approximately doubles in the course of the 11 cycles of the NI of the murine neocortical PVE. The histogenetic implications of the increase of TG1 across the NI are less clearly established than those of the increase in Q. Elsewhere we have suggested that the increase in TG1 with cycle may in some way not presently understood be related to mechanisms that regulate the transcriptional and translational profile necessary to specify both neocortical neuronal cell class and the regional neocortical protomap (Miyama et al., 1997Go; Caviness et al., 1999Go). These specification events appear to occur in the PVE coordinately with the proliferative process. They are indexed to cell cycle number and therefore to a specific range in duration of the G1 phase.

Variations in Expression of Cyclin E and p27 and Regulation of Q and TG1

The principal hypothesis arising from the present analysis, and implicit in Figures 5 and 6GoGo, is that the patterns of variation across the NI in the proliferative parameters Q and TG1 are regulated at a proximate molecular level by mechanisms which act, at least in part, through regulation of cyclin E and p27. This hypothesis is consistent with the established consequences of overexpression of cyclin E and p27 and with established roles of cyclin E, p27 and other proteins including p21 and cdk2 with which they interact in other proliferative vertebrate cell systems (Ohtsubo and Roberts, 1993Go; Fero et al., 1996Go; Kiyokawa et al., 1996Go; Nakayama et al., 1996Go). This hypothesis is to our knowledge unique in that it yokes the regulation of an entire vertebrate neuronogenetic sequence to the regulation of expression of proteins critical to cell cycle operation.

Cyclin E and p27 and Proliferative Behavior of Vertebrate Cells

Overexpression of cyclin E and p27 approximates the essential behaviors of proliferative cells, respectively, in phase 1 and phase 2 of the NI (Fig. 6Go). Under conditions of cyclin E overexpression in human diploid fibroblasts, most, if not all, postmitotic cells are driven through G1 phase into S phase (Ohtsubo and Roberts, 1993Go), and TG1 is greatly shortened. These behaviors are like those of the PVE in phase I of the neuronogenetic interval where Q is minimally greater than 0 and where TG1 increases only modestly from its minimum value.

Overexpression of p27, by contrast, induces postmitotic cells in G1 phase to leave the cycle rather than to re-enter S phase (Fero et al., 1996Go; Kiyokawa et al., 1996Go; Nakayama et al., 1996Go). The consequences of the value of TG1 for the residual P fraction, the fraction of postmitotic cells which remain in cycle, have not been worked out explicitly. However, the sharp elevation in cycle exit, i.e. the sharp elevation in the apparent value of Q, again catches the essential behavior of the PVE during phase II of the neocortical PVE. Consistent with the effect of overexpression of this protein, knockout of the p27 gene in mice results in an animal that is some 30–40% larger than animals with the wildtype gene. The enlargement is a uniformly scaled increase in body size and size of (most) internal organs, including the brain and even the individual laminae and neuronal populations of the neocortex, and occurs as a consequence of increase in cell number (Fero et al., 1996Go; Kiyokawa et al., 1996Go; Nakayama et al., 1996Go). By implication p27 may be inferred to be pervasively rate limiting for the proliferative process of all histogenetic systems (excepting a few lymphoid organs). It can be only partially rate limiting in its action; otherwise the animal would be not stop growing.

Regulation of Cyclin E and p27

The hypothesis presented here that the functions of cyclin E and p27, in conjunction with that of coordinately acting proteins, regulate the proliferative process of murine neocortical neuronogenesis is based upon a set of correlations arising out of observations which are for the present only descriptive in nature. This hypothesis is, however, experimentally testable. It is anticipated that the domain of regulation will be found to lie principally within the G1 phase of the cell cycle. This is because p27 and coordinately acting proteins probably govern Q by setting the probability that cells will exit the cell cycle as opposed to continuing to proliferate at the G1 restriction point. Cyclin E and coordinately acting proteins are expected to be found to act primarily at the G1/S transition. Increments in TG1 may be related in some way to execution of the transcriptional and translational cascade set in motion at the restriction point as the cascade increases in complexity over the NI (Caviness et al., 1999Go). In addition, work in dissociated non-neural vertebrate cell systems in vitro and from in vitro studies with explants or dissociated cells of the neocortical PVE itself implicate both cell external substances (Kilpatrick and Bartlett, 1993Go; Ghosh and Greenberg, 1995Go; LoTurco et al., 1995Go; Temple and Qian, 1995Go; Bonni et al., 1997Go; Cavanagh et al., 1997Go) and the operation of gap junctions as modulators of Q (LoTurco and Kriegstein, 1991Go; Bittman et al., 1997Go; Goto et al., 1998Go). We anticipate that this modulation will be reflected in regulated activity of p27 and, perhaps, cyclin E and that the capacity of the cell to respond to external influences is substantially developmentally regulated. We infer, therefore, that the inexorable advance of the proliferative program, manifested by systematic increases of Q and TG1, must reflect a coordinate but complex combination of cell internal regulatory mechanism and cell external modulators, and their balance is reflected in cyclin E and p27 mRNA levels.


    Acknowledgments
 
We gratefully acknowledge valuable suggestions and comments from Pradeep G. Bhide, Ed Harlow, Joriene de Nooij, Iswar Hariharan and Carrie G. Wager, and the expert technical assistance of Carol Hoover. Supported by NIH grants NS12005 and NS33433 and NASA grant NAG2–750. T.T. was supported by a fellowship of The Medical Foundation, Inc., Charles A. King Trust, Boston, MA.


    References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bayer SA, Altman J (1991) Neocortical development. New York: Raven Press.

Bittman K, Owens D, Kriegstein A, LoTurco J (1997) Cell coupling and uncoupling in the ventricular zone of developing neocortex. J Neurosci 17:7037–7044.[Abstract/Free Full Text]

Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank D, Rozovsky I, Stahl N, Yancopoulos G, Greenberg M (1997) Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 278:477–483.[Abstract/Free Full Text]

Cai L, Hayes N, Nowakowski R (1997) Local homogeneity of cell cycle length in developing mouse cortex. J Neurosci 17:2079–2087.[Abstract/Free Full Text]

Cavanagh J, Mione M, Pappas I, Parnavelas J (1997) Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters. Cereb Cortex 7:293–302.[Abstract]

Caviness VS Jr, Takahashi T, Nowakowski RS (1995) Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends Neurosci 18:379–383.[ISI][Medline]

Caviness VS Jr, Takahashi T, Nowakowski RS (1999) The g1 restriction point as critical regulator of neocortical neuronogenesis. J Neurochem Res 24:497–506.

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocynate–phenol–chloroform extraction. Anal Biochem 162:156–159.[ISI][Medline]

Delalle I, Bhide P, Caviness VSJr, Tsai L-H (1997) Temporal and spatial patterns of expression of p35, a regulatory subunit of cyclin dependent kinase 5, in the nervous system of the mouse. J Neurocytol 26:283–296.[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 85:733- 744.[ISI][Medline]

Ghosh A, Greenberg ME (1995) Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis. Neuron 15:89–103.[ISI][Medline]

Goto T, Takahashi T, Miyama T, Bhide P, Caviness VS Jr (1998) Gap junctions exert a developmentally regulated mitogenic effect upon neocortical proliferative epithelium. Soc Neurosci Abstr 24:280.

Halevy O, Novitch B, Spicer D, Skapek S, Rhee J, Hannon G, Beach D, Lassar A (1995) Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 267:1018–1021.[ISI][Medline]

Heintz N (1993) Cell death and the cell cycle: a relationship between transformation and neurodegeneration. Trends Biol Sci 18:157–159.

Hengst L, Reed SL (1996) Translational control of p27Kip1 accumulation during the cell cycle. Science 271:1861–1864.[Abstract]

Hunter T (1993) Braking the cycle. Cell 75:839–841.[ISI][Medline]

Kilpatrick TJ, Bartlett PF (1993) Cloning and growth of multipotential neural precursors: requirements for proliferation and differentiation. Neuron 10:255–265.[ISI][Medline]

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 85:721–732.[ISI][Medline]

Knoblich JA, Lehner CF (1993) Synergistic action of Drosophila cyclins A and B during the G2–M transition. EMBO J 12:65–74.[Abstract]

Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM (1992) Formation and activation of a cyclin E–cdk2 complex during the G1 phase of the human cell cycle. Science 257:1689–1694.[ISI][Medline]

Kornack DR, Rakic P (1992) Cell cycle kinetics of the ventricular zone in fetal monkey telencephalon. Soc Neurosci Abstr 18:30.

Kornack D, Rakic P (1998) Changes in cell-cycle kinetics during the development and evolution of primate neocortex. Proc Natl Acad Sci USA 95:1242–1246.[Abstract/Free Full Text]

LaBaer J, Garrett M, Stevenson L, Slingerland J, Sandhu C, Chou H, Harlow E (1996) New functional activities for the p21 family of Cdk inhibitors. Genes Dev 11:847–862.[Abstract]

Lee M-H, Nikolic M, Baptista C, Lai E, Tsai L-H, Massague J (1996) The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons. Proc Natl Acad Sci USA 93:3259–3263.[Abstract/Free Full Text]

Lew DJ, Dulic V, Reed SI (1991) Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell 66:1197–1206.[ISI][Medline]

LoTurco JJ, Kriegstein A (1991) Clusters of coupled neuroblasts in embryonic neocortex. Science 252:563–566.[ISI][Medline]

LoTurco JJ, Owens DF, Heath MJS, Davis MBE, Kriegstein AR (1995) GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15:1287–1298.[ISI][Medline]

Massague J, Polyak K (1995) Mammalian antiproliferative signals and their targets. Curr Opin Gen Dev 5:91–96.[Medline]

Miyama S, Takahashi T, Nowakowski RS, Caviness VSJr (1997) A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium. Cereb Cortex 7:678–689.[Abstract]

Morgan DO (1995) Principles of Cdk regulation. Nature 374:131–134.[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 85:707–720.[ISI][Medline]

Ohtsubo M, Roberts JM (1993) Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science 259:1908–1912.[ISI][Medline]

Pagano M, Tam S, Theodoras A, Beer-Romer P, Del Sal G, Chau V, Yew P, Draetta G, Rolfe M (1995) Role of the ubiquitin–proteasome pathway in regulating abundance of the cyclin- dependent kinase inhibitor p27. Science 269:682–685.[ISI][Medline]

Parker SB, Eichele G, Zhang P, Rawls A, Sands AT, Bradley A, Olson EN, Harper JW, Elledge SJ (1995) p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science 267:1024–1027.[ISI][Medline]

Polyak K, Lee M-H, Erdjument-Bromage H, Koff A, Roberts J, Tempst P, Massague J (1994) Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59–66.[ISI][Medline]

Sauer FC (1935) Mitosis in the neural tube. J Comp Neurol 62:377–405.

Sauer FC (1936) The interkinetic migration of embryonic epithelial nuclei. J Morphol 60:1–11.

Sheaff J, Groudine M, Gordon M, Roberts J, Clurman B (1997) Cyclin E-cdk2 is a regulator of p27Kip1. Genes Dev 11:1464–1478.[Abstract]

Sherr CJ (1994) G1 phase progression: cycling on cue. Cell 79:551–555.[ISI][Medline]

Sherr C (1996) Cancer cell cycles. Science 274:1672–1677.[Abstract/Free Full Text]

Sherr CJ, Roberts JM (1995) Inhibitors of mammalian G1 cyclin- dependent kinases. Genes Dev 9:1149–1163.[ISI][Medline]

Sidman RL, Miale IL, Feder N (1959) Cell proliferation and migration in the primitive ependymal zone: an autoradiographic study of histogenesis in the nervous system. Exp Neurol 1:322–333.[ISI]

Smart IHM, Smart M (1982a) Growth patterns in the lateral wall of the mouse telencephalon. I autoradiographic studies of the histogenesis of the iso-cortex and adjacent areas. J Anat 134:273- 298.[ISI][Medline]

Smart IHM, McSherry GM (1982b) Growth patterns in the lateral wall of the mouse telencephalon. II. Histological changes during and subsequent to the period of isocortical neuron production. J Anat 131:415–442.

Takahashi T, Nowakowski RS, Caviness VS Jr (1992) BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J Neurocytol 21:185–197.[ISI][Medline]

Takahashi T, Nowakowski RS, Caviness VS Jr (1993) Cell cycle parameters and patterns of nuclear movement in the neocortical proliferative zone of the fetal mouse. J Neurosci 13:820–833.[Abstract]

Takahashi T, Nowakowski RS, Caviness VS Jr (1995a) The cell cycle of the pseudostratified ventricular epithelium of the murine cerebral wall. J Neurosci 15:6046–6057.[Abstract]

Takahashi T, Nowakowski RS, Caviness VS Jr (1995b) Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall. J Neurosci 15:6058–6068.[Abstract]

Takahashi T, Goto T, Miyama S, Nowakowski RS, Caviness VS Jr (1996a) Intracortical distribution of a cohort of cells arising in the PVE. Soc Neurosci Abstr 22:284.

Takahashi T, Nowakowski R, Caviness VS Jr (1996b) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general computational model of neocortical neuronogenesis. J Neurosci 16:6183–6196.[Abstract/Free Full Text]

Takahashi T, Nowakowski RS, Caviness VSJr (1996c) Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. J Neurosci 16:5762–5776.[Abstract/Free Full Text]

Takahashi T, Nowakowski RS, Caviness VS Jr (1999) Cell cycle as operational unit of neocortical neuronogenesis. Neuroscientist (in press).

Temple S, Qian X (1995) bFGF, neurotrophins, and the control of cortical neurogenesis. Neuron 15:249–252.[ISI][Medline]

Tsai L-H, Lees E, Faha B, Harlow E, Riabowol K (1993a) The cdk2 kinase is required for the G1-to-S transition in mammalian cells. Oncogene 8:1593–1602.[ISI][Medline]

Tsai L-H, Takahashi T, Caviness VSJr, Harlow E (1993b) Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119:1029–1040.[Abstract/Free Full Text]

Vlach J, Hennecke S, Amati B (1997) Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27Kip1. EMBO J 16:5334–5344.[Abstract/Free Full Text]

Waechter RV, Jaensch B (1972) Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats. Brain Res 46:235–250.[ISI][Medline]

Xiong Y, Hannon G, Zhang H, Casso D, Kobayashi R, Beach D (1993) p21 is a universal inhibitor of cyclin kinases. Nature 366:701–704.[ISI][Medline]

Zhang H, Hannon G, Beach D (1994) p21-containing kinases exist in both active and inactive states. Genes Dev 8:1750–1758.[Abstract]