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Pax6 is required to regulate the cell cycle and the rate of progression from symmetrical to asymmetrical division in mammalian cortical progenitors

Guillermo Estivill-Torrus^1,*, Helen Pearson^2,*, Veronica van Heyningen², David J. Price^1, and Penny Rashbass^2,3,

¹ Department of Biomedical Sciences, University of Edinburgh Medical School, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK
² MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
³ Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
^* These authors contributed equally to the work

View larger version (90K): [in a new window]   Fig. 1. Counting parameters used for in vivo analysis. (A) Parasagittal view of an E12.5 wild-type forebrain showing the planes of section in which cell divisions were counted. Cells were counted in five sections at 25%, 50% and 75% through the telencephalon (broken lines). (B) Coronal section through an E12.5 wild-type embryo at a position 50% through the telencephalon (dorsal is upwards). Cell nuclei are stained with DAPI. Cell counts were made in the ventricular zone of the cortex between the two arrows. (C,D) Mitotic cells in (C) wild-type and (D) Pax6Sey/Sey E15.5 cortex identified with an antibody against phosphorylated histone H3, which labels metaphase chromosomes. In the wild-type cortex the majority of dividing cells are located at the ventricular surface; in the Pax6Sey/Sey cortex some dividing cells are located ectopically (away from the ventricular surface, arrows). (E) Late anaphase and telophase cells were classified by their plane of division relative to the ventricular surface (0°): –30° to 30°, asymmetrical division; 30° to 60°, intermediate division; 60° to 120°, symmetrical division. (F,G) DAPI-labelled chromosomes showing (F) symmetrical and (G) asymmetrical cell divisions. Broken line indicates plane of ventricular surface and arrow indicates plane of cell division. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Scale bars: 100 µm in B; 20 µm in C,D; 5 µm in F,G.

View larger version (10K): [in a new window]   Fig. 2. Analysis of ectopic cell division and distribution of cleavage orientation in wild-type and Pax6Sey/Sey cortex at E10.5, E12.5 and E15.5. (A) Histogram showing the mean percentage (±s.e.m.) of late anaphase and telophase cells that are dividing in an ectopic postion (away from the ventricular surface) in wild-type cortex and Pax6Sey/Sey cortex. There is an increase in ectopic division at E15.5 in the mutants(P<0.0005). (B,C) Graphs comparing the mean percentages of dividing cells undergoing symmetrical, asymmetrical and intermediate division in (B) wild-type and (C) Pax6Sey/Sey cortex. All data points are ±2%.

View larger version (16K): [in a new window]   Fig. 3. Analysis of cell-cycle kinetics. (A,B) Graphs show results of cumulative labelling with BrdU at (A) E12.5 and (B) E15.5 in Pax6Sey/Sey and wild-type embryos, following the approach described by Nowakowski et al. (Nowakowski et al., 1989). Data points are means±s.e.m. of the proportions of BrdU-labelled cells in the cortex of each embryo. The proportions increased over 4.5 hours in E12.5 Pax6Sey/Sey embryos (broken line in A) and over 8-8.5 hours in all other cases. Least squares fit analysis of these rising phases (i.e. excluding data after giving 8.5 hours of BrdU to Pax6Sey/Sey embryos aged E12.5 and after giving 12.5 hours of BrdU to embryos of both genotypes aged E15.5) yielded the trendlines and r2 values shown. (C) The growth fraction (GF), the length of S phase (Ts) and the length of the cell cycle (Tc) are calculated using the equations of Nowakowski et al. (Nowakowski et al., 1989). The intercept on the y-axis=GFx(Ts/Tc); the time where the GF is maximal (Tm)=Tc-Ts; the rate of increase (i.e. the slope) of the proportion of labelled cells=GF/Tc. (D) Cell-cycle kinetics in wild-type and mutant telencephalon at E12.5 and E15.5. The circumference of each circle is proportional to the overall length of the cell cycle at the different stages. The wild-type cell cycle lengthens slightly between E12.5 and E15.5. In the mutant, overall cell-cycle length is significantly shorter than normal at E12.5 and longer than normal at E15.5. The proportion of time that the cells spend in S phase is not significantly different between mutant cortex and wild-type cortex at E12.5. However, by E15.5 the proportion of time in S phase is markedly increased in the mutant.

View larger version (107K): [in a new window]   Fig. 4. Expression of BrdU and TuJ1 in wild-type and Pax6Sey/Sey E12.5 cortex. Parasagittal sections through cortex of (A) wild-type and (B) Pax6Sey/Sey E12.5 telencephalon. Dams were injected at 0 and 2 hours and embryos collected at 2.5 hours after initial injection. Broken line indicates boundary of VZ. There is a clear increase in double-labelled cells (BrdU, brown; TuJ1, purple) in the mutant (B) compared with the wild-type (A) littermate. Scale bar: 60 µm.

View larger version (99K): [in a new window]   Fig. 5. Expression of Pax6 and Numblike (Nbl) in wild-type and Pax6Sey/Sey cortex. In all sections dorsal is upwards and in colour images nuclei are counterstained with DAPI (blue). (A-F) Coronal sections through (A-C) E10.5 wild-type and (D-F) E10.5 Pax6Sey/Sey brains. (G-L) Parasagittal sections through (G-I) E15.5 wild-type and (J-L) E15.5 Pax6Sey/Sey brains. (A,D,G,J) Low-magnification images of (A,G) wild-type and (D,J) Pax6Sey/Sey brain sections stained with DAPI. Boxes show areas in the cortex in which images to right were taken. (B,E,H,K) Nuclear Pax6 expression (green) is detected in wild-type ventricular zone (B,H) but not in the mutant (E,K). (C,F,I,L) Weak cytoplasmic Nbl expression (green) is detected in a few cells in E10.5 wild-type preplate (PP) (C). The Nbl expression domain has expanded in the wild-type cortex by E15.5 (I): expression is observed in both the wild-type intermediate zone (IZ) and cortical plate (CP), although expression levels appear to be much stronger in the IZ compared with the CP. (F,L) In the mutant, more Nbl-positive cells are detected in the preplate at E10.5 (F) compared with wild type (C). Nbl is highly expressed by cells in the Pax6Sey/Sey IZ and CP at E15.5 (L). The Nbl expression appears higher in the mutant CP compared with wild-type levels. c, cerebral vesicle; d, diencephalon; SVZ, subventricular zone, VZ, ventricular zone. Scale bars: 100 µm in A,D; 10 µm in B,C,E,F,H,I,K,L; 200 µm in G,J.

View larger version (79K): [in a new window]   Fig. 6. Immunocytochemical analysis of dissociated cortical cells in culture. (A,C,E,G,I) Wild-type and (B,D,F,H,J) Pax6Sey/Sey dissociated cortical cells after 12.75 hours (E-J) and 24 hours (A-D) in culture. (A-D) BrdU labelling in cultures 24 hours after plating (11.25 hours after the end of the BrdU pulse). (A,B) Double immunostaining for BrdU (brown) and MAP2 (blue). In contrast to the wild-type BrdU-labelled cells, Pax6Sey/Sey-labelled cells were mainly in large clusters. (C,D) Many mutant BrdU-labelled cells were grouped as to suggest cell-chain migration. Inset in D shows a panoramic view of mutant cortical cell-chain formation in culture. (E-J) Immunodetection of (E,F) neuronal antigen class III ß tubulin (TuJ1), (G,H) nestin and (I,J) MAP2 (blue) and RC2 (brown). Arrowheads in I,J show cells expressing both markers. Scale bars: 24 µm in A,B,E-J; 16 µm in C,D.

View larger version (27K): [in a new window]   Fig. 7. Proliferation and differentiation in E12.5 wild-type and mutant dissociated cortical cell cultures between 12.75 and 24 hours after plating (all values are means±s.e.m.). (A) Percentages of BrdU-positive cells after a 45 minute pulse given 12 hours after plating. Significant differences (P<0.01) were observed between cultures of wild-type and mutant cells at all time-points from 12.75 hours after plating. (B) Cell density in cultures. Significant differences (P<0.01) were observed between cultures of wild-type and mutant cells at all time-points from 12.75 hours after plating. Regression analysis showed that the rate of increase for the mutant cells was just over twice that for the wild-type cells (r2=0.88 for Pax6Sey/Sey and 0.99 for wild type). (C,D) Percentages of cells expressing cyclin A (C) and p27kip1 (D). Wild-type cells show no significant change in the number of cells expressing cyclin A and p27kip1 over the culture period (r2=0.07 for wild-type data in C and 0.22 for wild-type data in D). There is a significant decrease in the percentage of mutant cells expressing cyclin A in culture (r2=0.84). The percentages of Pax6Sey/Sey cells expressing p27kip1 increase with time (r2=0.99).

View larger version (8K): [in a new window]   Fig. 8. Explanation of the most probable cause of the differences observed in the cultures (Fig. 7). (A) In wild-type cells, the data are compatible with cells having a cell-cycle time of around 12 hours, as has been shown previously in vivo (Takahashi et al., 1995b) (present study). Some cells are in S-phase during the 45 minute BrdU pulse and incorporate BrdU (filled circle); others will enter S-phase after the BrdU pulse or are postmitotic and will not label with BrdU (open circles). One in five cells are BrdU labelled following the pulse (i.e. 20%; compare with wild-type data at 12.75 hours in Fig. 7A). As the cells containing BrdU divide (thick lines), the proportion of BrdU-labelled cells in the culture increases. Once the nonlabelled cells divide (thin lines), the proportion of BrdU-labelled cells will decrease again. This trend is seen for wild-type data in Fig. 7A. Broken lines indicate cells that are postmitotic. Three in five cells are postmitotic at the time of the BrdU pulse (i.e. 60%; compare with wild-type data at 12.75 hours in Fig. 7D) and if half the divisions that occur in culture produce postmitotic neurones (shown as a broken line originating from the nonlabelled cell, although it could originate from the labelled cell), then roughly the same number will be postmitotic at 24 hours (four in seven cells, i.e. 57%; compare with wild-type data at 24 hours in Fig. 7D). The proportions of postmitotic cells and progenitor cells will not change greatly during culture in the scheme illustrated. (B) An interpretation of events in cultures of Pax6Sey/Sey cells is shown, using the same conventions as in A. A shorter cell cycle produces an initially higher proportion of BrdU-labelled cells (filled circles; two in five cells, or 40%; compare with data from mutants in Fig. 7A) and a subsequent steady increase in these proportions (four in seven cells at 18 hours; six in ten cells at 24 hours). This models the steady increase seen for mutant cells in Fig. 7A. The scheme illustrated, in which divisions produce one progenitor and one postmitotic cell, will lead to an abnormally rapid decrease in the proportions of progenitors and an increase in the proportions of postmitotic cells (from 40% at the time of the BrdU pulse to 70% at 24 hours; comparable with data from mutants in Fig. 7D).