Time course of the distribution of AO-stained apoptotic cells in the irradiated brains of wt and p53^–/–embryos.

<p>Wt (A, B, C, D) and p53^{<b>–/–</b>}embryos (E, F, G, H) were irradiated with 10 Gy of gamma rays and stained with AO at 3 h (A, E), 12 h (B, F), 24 h (C, G), and 42 h (D, H) after irradiation. Images of clusters of AO-positive spots at higher magnifications (white arrows in squares in B and F) are shown in boxes (I and J) for detailed views of the rosette-shaped clusters of apoptotic neurons, which were fewer and smaller in the OT of p53^{<b>–/–</b>}embryos. Also shown are a bright-field counterpart image for AO-stained fluorescence images (K) and a schematic diagram illustrating the structure of the embryonic medaka brain at stage 30 (L). CE, cerebellum; OT, optic tectum; TE, telencephalon. Scale bars = 50 μm.</p

Time course of the numbers of AO-positive rosette-shaped clusters in the OT of irradiated wt and p53^–/–embryos.

<p>The numbers of AO-positive rosette-shaped clusters in the OT were counted at various times after gamma-ray irradiation (10 Gy). Error bars show the SEM (<i>n</i> = 3). Statistical differences between the means for wt (solid line) and p53^{<b>–/–</b>}embryos (broken line) were evaluated using Student’s unpaired <i>t</i> tests after <i>F</i> tests. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

The number of radiation-induced apoptotic cells drastically decreased as embryogenesis proceeded.

<p>Embryos at 3, 4 and 5 dpf were irradiated with 15 Gy of gamma rays, and irradiation-induced apoptotic cells in the brain (square areas in A and E) were examined by light microscopy (B–D) and AO assay (F–K) at 24 h after irradiation. Irradiation-induced apoptotic cells in the trunk (rectangular area in E) were examined by AO-assay (L–Q). Opaque dead cells were identified in the marginal area of the OT in irradiated 3 dpf (arrows in B) and 4 dpf (arrows in C) embryonic brains. In irradiated 5 dpf embryonic brain, no opaque dead cells were evident (D). The distribution of AO-stained apoptotic cells in the brain of irradiated 3–5 dpf embryos (arrows in F, G) and in the trunk of irradiated 3–5 dpf embryos (arrows in L, M) were similar manner to those by microscopic observations (arrows in B, C), whereas no apoptotic cells were found in the brain and trunk of sham controls (I-K and O-Q), respectively. Scale bars = 50 μm.</p

Immuno-histological analyses of irradiation-induced apoptosis in mature proliferating tissues of adult medaka.

<p>(A) The sagittal section of whole-body adult medaka as a high-resolution seamless tiling image was cited from a web browser (<a href="https://ds.cc.yamaguchi-u.ac.jp/~vs_08_2p/newpage4.html" target="_blank">https://ds.cc.yamaguchi-u.ac.jp/~vs_08_2p/newpage4.html</a>; image from HdrR_M_221.). In mature medaka, actively proliferating tissues including pharyngeal epithelium, small intestine and testis (squared area with dotted outline in A) were examined 24 h after irradiation. PCNA-positive cells were present in a proliferating area of the pharyngeal epithelium (arrows in C), the bottom of intestinal folds (arrows in E) and in cysts of proliferating spermatogonial cells (Gb) in the testicular tissue (arrows in G). In contrast, no cleaved-caspase 3-positive signals were observed 24 h after irradiation in the pharyngeal epithelium (B) and the intestinal tissue (D). In the mature testis, many cleaved-caspase 3-positive cells were present in the Gb cysts (arrows in F) where PCNA-positive proliferating spermatogonial cells are localized (arrows in G), whereas no cleaved caspase 3 -positive cells were found in the Ga cysts, circled area with black dotted outline in F, where no PCNA positive cells were present (circled areas with black dotted outline in G). Scale bars = 50 μm.</p

Histological analyses of irradiation-induced apoptotic cells in the pharyngeal epithelium during later period of embryogenesis.

<p>(G) shows the image of alizarin red stained embryo at hatching period. Frontal cryo-sections were prepared at the level of the solid line in G (A–F). Immunohistochemical analyses of pharyngeal epithelium tissue (squared area in H) were performed 24 h after irradiation with 15 Gy of gamma rays. Many cleaved-caspase 3-positive cells were present in irradiated 3 dpf embryos (arrow in A), thereafter these signals were relatively fewer in irradiated 4 dpf embryos (arrow in B). No positive signal was evident in irradiated 5 dpf embryos (C). Highly proliferative cells, corresponding to anti-P-H3-positive signals were present in the pharyngeal epithelium of normal development during later embryogenesis, at 4 dpf (D), 5 dpf (E) and 6 dpf (F). Scale bars = 50 μm.</p

Histological analyses of the time course of neuronal damage in irradiated wt brains.

<p>Wt medaka embryos were irradiated with 10 Gy of gamma rays. Frontal sections of plastic-embedded heads were prepared in the plane that included their eyes and subjected to Nissl staining with cresyl violet. Clusters of apoptotic nuclei are shown in the irradiated wt embryos at 12 h (arrowhead in A), 24 h (arrowhead in D), and 42 h after irradiation (arrowhead in G); images at higher magnification in squares with dotted outlines in A, D, and G are shown with arrowheads in B, E, and H, respectively. Scale bars = 50 μm. Electron microscopic images of apoptotic clusters in the dotted-line boxes in B, E, and H are shown in C, F, and I, respectively. Microglia engulfed 10–15 apoptotic neurons into their phagosomes, and the nuclei of the engulfed apoptotic neurons maintained their appearance almost intact to 12 h after irradiation (C). The nuclei of the phagocytosed apoptotic neurons gradually became fragmented during the following 12 h (F). At 42 h after irradiation, degradation of apoptotic nuclei in phagosomes was almost complete (I). Frontal plastic sections (A, B, D, E, G, and H) were prepared at the level of the solid line of the embryonic brain shown in (J). CE, cerebellum; OT, optic tectum; MES, mesencephalon; TE, telencephalon. Scale bars in A, B, D, E, G, and H = 50 μm. Scale bars in C, F, and I = 2 μm.</p

Histological analyses of irradiation-induced apoptotic cells in the brains and eyes during later period of embryogenesis.

<p>(A) shows schematic image of embryonic head. Frontal plastic sections (C–N) and cryo-sections (O–T) of the embryonic brain were prepared at the level of the solid line shown in A. Apoptotic cells were examined in the brain and eye (squared area with dotted line in B). Nissl-stained sections of embryos 24 h after irradiation of 3 dpf (C, I) 4 dpf (D, J) and 5 dpf (E, K) and respective controls (F, L), (G, M), and (H, N). Immunohistochemical sections with anti-cleaved-caspase3 of embryos 24 h after irradiation of 3 dpf (O) 4 dpf (Q) and 5 dpf (S). Magnified images in squared area with dotted outlines in O, Q and S are shown in P, R and T, respectively. In irradiated 3 dpf embryos, pyknotic cells were present in the marginal area of the OT (arrow in C) and in the eye (arrowheads in I) and cleaved-caspase 3-positive apoptotic neurons are present in the brain (arrows in O and P) and eyes (arrowheads in O). Fewer apoptotic cells were found in the brain (arrows in D, Q and R) and eyes (arrowheads in J and Q) of irradiated 4 dpf embryos, and no apoptotic cells were found in irradiated 5 dpf embryos (E, K, S and T). No pyknotic cells were present in the brain and eyes of sham controls of 4–6 dpf embryos (F–H and L–N), respectively. Scale bars = 50 μm.</p

Distribution of ApoE-expressing microglia during phagocytosis shown by WISH.

<p>Activated microglia were identified as ApoE-expressing cells by WISH in nonirradiated control embryos (A), irradiated embryos at 24 h after irradiation (E), and at 42 h after irradiation (J). Frontal plastic sections including the eyes and the OT at the ‘a’ and ‘b’ levels of the brain in A, E, and J are shown in B, F, and K, and C, G, and L, respectively. A few ApoE-expressing cells were present in the retina of wt nonirradiated embryos (arrowhead in C), in the TE (arrowheads in B), and the OT (arrowhead in C). At 24 h after irradiation, hypertrophic and rounded ApoE-expressing microglia (H) appeared in the TE (arrowhead in F), retina (arrowhead in F, G), and OT (arrowhead in G). Unstained round areas were present in the TE, retina, and marginal regions of the OT (open arrowheads in F and G). At 42 h after irradiation, the number of ApoE-expressing microglia had increased markedly (arrowheads in K and L) and they showed a branched morphology (M). The numbers of unstained areas in the TE and OT (open arrowheads in K and L) decreased and they were small, not hypertrophied. Three-dimensional images were constructed from serial sections of WISH-stained nonirradiated (D), embryos at 24 h (I) and at 42 h after irradiation (N). ApoE-expressing microglia are in red and the unstained round areas appear white. MES, mesencephalon; OT, optic tectum; TE, telencephalon. Scale bars = 50 μm.</p

Increased numbers of ApoE-expressing cells were present in p53^-/- embryos at the late phase of phagocytosis.

<p>The p53^{<b>–/–</b>}embryos were irradiated with 10 Gy of gamma rays and frontal sections of heads including the eyes embedded in plastic resin were cut at the level of the solid line in A and subjected to Nissl staining with cresyl violet. Clusters of apoptotic nuclei were present at 24 h after irradiation (arrowhead in B) and images at higher magnification of the squares with dotted outlines in B are shown in C with an arrowhead. An EM image of phagocytosed apoptotic nuclei in a microglial phagosome (D) showed complete digestion of apoptotic nuclei in microglial phagosome as in wt embryos at 42 h after irradiation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127325#pone.0127325.g003" target="_blank">Fig 3I</a>). Activated microglia were identified as ApoE-expressing cells by WISH in irradiated p53^{<b>–/—</b>}embryos 24 h after irradiation (E). Frontal plastic sections including the eyes and the OT of WISH-stained p53^{<b>–/–</b>}embryos (stage 30) at the ‘a’ and ‘b’ levels of the brain in E are shown in F and G, respectively. The numbers of ApoE-expressing microglia increased (arrowheads in F and G) and they showed a branched morphology, not hypertrophy (a higher magnification is shown in H). The 3D reconstructed images showed dramatically increased numbers of ApoE-expressing cells in the irradiated p53^{<b>–/–</b>}embryos (I), as in wt embryos 42 h after irradiation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127325#pone.0127325.g004" target="_blank">Fig 4N</a>). MES, mesencephalon; OT, optic tectum; TE, telencephalon. Scale bar in (D) = 2 μm; Scale bars in (B–G) = 50 μm.</p

Radical change of apoptotic strategy following irradiation during later period of embryogenesis in medaka (<i>Oryzias latipes</i>)

<div><p>Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.</p></div