Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly

Abstract

The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly.

Fig 1. IR induces microcephaly in mice.

(A) Mouse embryos at E13.5 were exposed in utero to IR of 1 or 2 Gy, and then embryonic brains were sampled at the indicated days. (B) Embryonic brains were weighed and average weights were calculated using at least 3 embryos. Error bars indicate the standard error. (C) Mouse embryos at E13.5 were exposed to 1 or 2 Gy, and then the brains of newborn mice (P0.5) were cryosectioned and stained with antibodies against Brn1 (green) and Foxp2 (red), used as markers of cerebral cortex layers II–IV and layer VI, respectively. Scale bar: 100 μm.

Fig 2. Apoptotic cell death in the mouse embryonic cerebral cortex after IR exposure.

(A) Embryonic brains were sampled at 24 h after 1 or 2 Gy IR exposure and stained with antibodies against cleaved-caspase-3 (apoptosis marker) and βIII-tubulin (Tuj1; neuron marker). (B) and (C) Cleaved-caspase-3-positive cells after 1 or 2 Gy exposure were quantified. Error bars indicate the standard error. (D) Embryonic brains were sampled at 24 h after 1 or 2 Gy exposure and stained with a Tbr2 antibody (neural-progenitor marker), and then the Tbr2-positive cells were quantified. At least 3 independent mouse embryos were used for each experiment. V: ventricle; VZ: ventricular zone; SVZ: subventricular zone. The border of VZ and SVZ was delineated according to the method by Pulvers JN., et al [12]. Scale bar: 100 μm.

Fig 3. Disruption of the apical layer in the mouse embryonic cerebral cortex after IR exposure.

Mouse embryos at E13.5 were irradiated with 1 or 2 Gy and the brains were sampled 24 h later and stained with antibodies against (A) ZO1 (green) and Sox2 (blue), used as markers of adherence junctions and neural progenitors, respectively; and (B) γ-tubulin (green), used as a spindle marker. VZ: ventricular zone; SVZ: subventricular zone. Scale bar: 100 μm.

Fig 4. Cell proliferation in the mouse embryonic cerebral cortex after IR exposure.

(A) Mouse embryos at E13.5 were irradiated with 1 or 2 Gy, and 24 h later, brains were sampled and stained with an antibody against Ser10-phosphorylated histone H3 (pH3; red, mitotic-cell marker) and the Tuj1 antibody (green, neuron marker), and counterstained with DAPI (white). Apical and basal mitotic cells are indicated by white and yellow arrows, respectively. (B) and (C) All pH3-positive cells in the apical and basal surface were quantified separately after irradiation with 1 Gy (B) or 2 Gy (C). Black and white bars indicate the apical and the basal surface, respectively. At least 3 independent mouse embryos were used for each experiment. Error bars represent the standard error. V: ventricle; VZ: ventricular zone; SVZ: subventricular zone. Scale bar: 100 μm.

Fig 5. Abnormal cytokinesis in the mouse embryonic cerebral cortex after IR exposure.

(A) Mouse embryos at E13.5 were irradiated with 1 or 2 Gy, and 48 h later, brains were sampled and stained with antibodies against Ser10-phosphorylated histone H3 (pH3; red in merged image) and γ-tubulin (green in merged image), used as markers of mitotic cells and centrosomes. Cells were counterstained with DAPI (blue in merged image). Quantification of (B) multipolar cell division, (C) lagging chromosomes, and (D) centrosome clustering among pH3-positive cells; at least 100 pH3-positive cells were counted. Error bars represent the standard error. Scale bar: 10 μm.