G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex

Abstract

We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.

Figure 1. Compartmentation of the primate germinal zones

A 5 micron thick Nissl stained parasaggital sections cutting through area 17 and area 18 germinal zones at E64. B 5 micron thick Nissl stained parasaggital sections cutting through area 17 and area 18 germinal zones at E78. C H3 immunolabelling showing the location of mitosis in area 17 germinal zones at E78. Abventricular mitosis are observed in the ISVZ and OSVZ. Adjacent microphotograph shows fluorescent nuclei staining with Propidium Iodide. D H3 immunolabelling showing the location of mitosis in area 18 germinal zones at E78. Adjacent microphotograph shows fluorescent nuclei staining with Propidium Iodide. E 5 micron thick eosin-haematoxyllin stained section of the germinal zones of area 17 at E64. Mitotic figures are observed in the ISVZ and OSVZ as indicated on the chart. F High power microphotograph showing abventricular mitosis from the insert in panel E. Abbreviations, CP cortical plate, IFL inner fiber layer, ISVZ inner subventricular zone, MZ marginal zone, OFL outer fiber layer, OSVZ outer subventricular zone, SP subplate, VZ ventricular zone, WM white matter. Scale bars: A to E : 100 microns, F : 10 microns.

Figure 2. Germinal origin of cortical neurons

A Area 17, E64 H3-thymidine pulse, three-hour survival. B Area 17, E78 H3-thymidine pulse, one-hour survival. C Area 17, E78 H3-thymidine pulse, 87-day survival. D Distribution of labeled precursors in OSVZ and VZ at E64, E78 and E89. Abbreviations as in Fig 1. Scale bars: 100 microns.

Figure 3. Cell-cycle kinetics of area 17 and 18 precursors

A Microphotograph of E78 area 17 dissociated precursors. MAP2 (red) and PCNA (brown) immunolabeling at 4 DIV. Scale bar: 20 microns. B,C,D in vitro values B cell density (CD) values at E78. C. LI values at E62 after 3DIV D LI values at E78 after 3DIV. Values ± SEM. Statistical significance: t test. E Cartoon illustrating principles of 3H-Thy cumulative labeling. This technique is based on a continuous 3H-Thymidine exposure that leads to the incorporation of 3H-Thymidine by successive cohorts of cycling cells progressing through S phase. The projection of the LI = 100% on the x-axis gives Tc- Ts. Ts is given by the projection of LI = 0 on the x-axis. F 3H-Thy cumulative labeling at E78 in vivo. Logistic regression combined with a X2 analysis shows the two slopes are significantly different. G Cartoon showing principles of PLM (Percentage of labeled mitotic figures). This procedure, based on a brief exposure of cells to 3H-Thymidine, measures the time required for cells in S phase to enter M phase and therefore returns Tg2/m. H PLM values at E78. Statistical analysis (F test) shows the slopes are identical. I Percentage of mitoses in area 17 and area 18 OSVZ at E78. Values are ± SEM.

Figure 4. Tissular and cellular levels of expression of Cyclin E and p27^Kip1

A Cyclin E, p27^Kip1, immunolabelling and Syto 16 counterstaining in area 17 germinal zones at E80. B Cyclin E, p27^Kip1, immunolabelling and Syto 16 counterstaining in area 18 germinal zones at E80. C upper panel: high magnification of Cyclin E positive precursors in the OSVZ, taken from A; lower panel: high magnification of OSVZ precursors counterstained with Syto 16, showing that only a fraction of precursors express high levels of Cyclin E. D Real Time PCR analysis of Cyclin E mRNA levels at E82. E Tissular Cyclin E expression levels (confocal quantification of Cyclin E fluorescent immunolabeling corrected for cell density in the OSVZ). F Tissular p27^Kip1 expression in area 17 and area 18 OSVZ (confocal quantification of p27^Kip1 fluorescent immunolabeling corrected for cell density in the full-width of the germinal zone). G Proportions of cells with high, medium and low Cyclin E levels in area 17 and area 18 OSVZ. H Confocal image of PCNA nuclear immunostaining on dissociated precursors from area 17 after 2 DIV. I Confocal image of p27^Kip1 immunolabeling (same field of view as H). J p27^Kip1 cellular expression levels in PCNA positive cells after 2 DIV. p27^Kip1 levels of expression are significantly lower in area 17 precursors than in area 18 precursors. Scale bars A, B: 100 microns ;C:25 microns ; H, I: 50 microns. Abbreviations see Figure 1.

Figure 5. Frequency of cell-cycle re-entry

A Cartoon showing how Cyclin E informs on frequency of cell-cycle re-entry. The G1 phase is divided into early and late G1 separated by the restriction point (R). The restriction point corresponds to the time point where a cell is irreversibly committed to a new cycle. Molecules expressed after the restriction point distinguish between daughter cells that re-enter the cycle from those that exit the cycle. Cyclin E expression characterizes late G1 and is therefore only expressed by daughter cells that re-enter the cell cycle. Hence, changes in proportions of precursors that express Cyclin E reflect variations in proportions of cells re-entering the cell-cycle. B In vivo percentage of Cyclin E-positive precursors in area 17 and area 18 OSVZ with respect to the total population of precursors. Values are ± SEM. Statistical analysis with Mann-Whitney U test; ***p<0.0005.

Figure 6. Analysis of cell-cycle parameters in p27^Kip1 and Cyclin E loss and gain of function experiments

A Drawing showing a lipofected precursor in an organotypic slice. B Microphotograph showing the GFP lipofected precursor from A in the OSVZ. C Microphotograph of a Ki67 positive, GFP lipofected precursor. D Histograms showing the percentage variation of cell-cycle re-entry (fraction of Ki67 positive cells in the germinal zones) value on organotypic slices. Control values correspond to precursors colipofected with control (pHPCAG) and EGFP plasmid or precursors colipofected with a nonsense control siRNA and EGFP plasmid E Histograms showing LI values on dissociated cultures. Control values correspond to precursors colipofected with control (pHPCAG) and EGFP plasmid. Statistical significance with a χ² test. F LI variations in area 17 and area 18 OSVZ precursors infected with Adnul. Results showing variations of LI (G), GF (H) and CD (cell density, I) in area 17 precursors overexpressing p27^Kip1 via adenoviral infection compared to area 18 precursors infected by empty virus. Scale bars B: 50 microns ; C: 10 microns. Abbreviations see Figure 2.

Figure 7. Mathematical modeling showing the evolution of the size of the precursor pool and total number of postmitotic neurons produced during the 15 day simulation period

Initial number of precursors (P) is identical in each case and arbitrary fixed at 100 (see text). Simulation is using in vivo Tc values obtained in this study (A17: 36 hours, A18: 46 hours) A Here, the frequency of cell-cycle re-entry is made identical for areas 17 and 18. The simulation shows that cell-cycle duration differences alone generate only a 14% difference in the number of neurons produced (N). B Here, Tc is fixed at 46 hours in both sets of precursors. Frequency of cell-cycle re-entry is set 20% higher in area 17 compared to area 18 (calculated from data of Figure 6, and corrected for variations in Tc). The simulation shows that cell-cycle re-entry difference alone generates a 75% difference in the number of neurons produced. C This simulation combines the experimentally observed a 22% difference in cell-cycle duration (36 vs 46 hours) and the observed 20% difference in cell-cycle re-entry (after correction for Tc). This generates a 151 % difference in the number of neurons produced.