Apoptosis and its relation to the cell cycle in the developing cerebral cortex - PubMed (original) (raw)

Apoptosis and its relation to the cell cycle in the developing cerebral cortex

D Thomaidou et al. J Neurosci. 1997.

Abstract

Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre- and early postnatal development of the rat cerebral cortex using in situ end labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [3H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.

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Figures

Fig. 1.

Apoptotic cells in the proliferative zones of the developing cortex. A, These cells (arrows) are distinguished by intense blue staining of the condensed nucleus in toluidine blue-stained semithin sections through the SVZ of an E19 rat. B, TUNEL-positive cells in the SVZ of a newborn rat revealed immunohistochemically using streptavidin Texas Red as a second layer. C, A TUNEL-positive cell in the SVZ of a newborn rat, using streptavidin peroxidase as a secondary antibody. LV, Lateral ventricle. D, Ultrathin section through the VZ of an E16 rat showing a cell with typical morphological characteristics of apoptosis. Magnifications: A–C, 640×;D, 11,700×.

Fig. 2.

Camera lucida drawings of coronal sections through E14 (A), E16 (B), E19 (C), P0 (D), P7 (E), and P14 (F) rat brains, illustrating the positions of dying cells, as revealed by TUNEL immunohistochemistry in 10-μm-thick paraffin sections. CP, Cortical plate;IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone. Calibration bars: A–C and_D–F_, 200 μm.

Fig. 3.

Illustrations of the methods used for the evaluation of the length of the cell cycle in the SVZ of newborn rats.A, Cumulative BrdU labeling indicating the maximum number of dividing cells after multiple BrdU injections.LV, Lateral ventricle. B, Toluidine blue staining of a semithin section through the SVZ, where mitotic figures are indicated by arrows. C, Adjacent section to the previous one, stained with anti-BrdU monoclonal antibody, showing labeled (thin double arrows) and unlabeled mitoses (thick arrows).Asterisks mark the same blood vessels in consecutive sections. Magnification: A, 110×; B,C, 640×.

Fig. 7.

A, Confocal image showing colocalization (yellow) of TUNEL-positive cells (red) with BrdU (green) after 15.5 hr of BrdU cumulative labeling. Note that not all TUNEL-positive cells are dividing. B, Confocal image showing a number of labeled SVZ cells: BrdU-labeled (green), TUNEL-positive (red), double-labeled (BrdU–TUNEL,yellow; BrdU–[3H] thymidine,green with blue grains), and triple-labeled cells (TUNEL–BrdU–[3H]-thymidine,yellow with blue grains). Magnification, 560×.

Fig. 4.

A, The percentage of labeled mitoses method was used to determine the length of_T_C and _T_G2+M of newborn SVZ cells. The results shown arise from two separate sets of experiments, one in paraffin sections and the other in semithin Araldite sections. After a pulse of BrdU at time 0, labeled mitoses were seen first after 2 hr, and the number increased rapidly to 100%. From this initial slope, the length of G2+M can be estimated as the interval between the injection and the time when labeling reached 50%, i.e., 3 hr. The number of labeled mitoses increased again as daughters of the initially labeled cells began the second round of division. The interval between the 50% points of the two successive ascending curves represents the length of _T_C and corresponds to ∼17 hr. B, Cumulative BrdU labeling of newborn SVZ cells. Each data point represents the mean ± SEM of counts obtained from five different animals. From an extrapolation of a linear repression line drawn through the initial ascending curve, the percentage of cells labeled at time 0 (LI0) was 15.7%, and the time taken to reach the plateau was 13.5 hr (_T_C −_T_S). From these data,_T_C was calculated as 18.6 hr and_T_S as 5.1 hr.

Fig. 5.

Schematic representation of the experimental procedure used to mark a cohort of dividing cells and trace them throughout the cell cycle. Open circles, BrdU-labeled cells; gray circles, BrdU and [3H]-thymidine-labeled cells; black circles, BrdU and TUNEL-positive cells. A, Rats received a single injection of BrdU at 0 min (open arrow) that marked all cells passing through S phase at that time. B, After 40 min, rats received an injection of [3H]-thymidine (black arrow), which also labeled cells passing through S phase. Only a cohort of cells that had exited the S phase during the last 40 min and were BrdU-positive, but [3H]-thymidine-negative (open circles), was followed. C, At 2 hr after the first injection, these cells were in mitosis and showed no sign of apoptosis, as indicated using TUNEL immunohistochemistry. D, At 4 hr after the first injection, these cells were in early G1 and again showed no sign of apoptosis. E, BrdU-positive–[3H]-thymidine-negative cells died during a narrow time window 5–8 hr after the injection of BrdU and were not seen to die at a later time within G1 (F).

Fig. 6.

The percentage of labeled mitoses method also was used to evaluate the length of M phase for cycling newborn SVZ cells. An identifiable cohort of cells moving synchronously along the cell cycle entered G1 phase 1 hr and 50 min after the injection of the first S-phase marker. Mitoses labeled only with this marker were visible for a total of 1.5 hr. After subtracting the length between injections of the two S-phase markers, the length of M phase was estimated to be no more than 50 min. Each point represents the mean ± SEM of counts in three different animals.

Fig. 8.

Agarose gel showing DNA fragmentation in the SVZ.Lane 1, 100 bp DNA ladder; lane 2, DNA from cultured thymocytes treated with dexamethasone; lane 3, DNA extracted from the SVZ of newborn rats showing a characteristic pattern of DNA fragmentation; lane 4, DNA extracted from adult cerebral cortex, used as a negative control of apoptosis.

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Apoptosis and its relation to the cell cycle in the developing cerebral cortex - PubMed (original) (raw)