Fig 1.
Cell type specific accumulation of γ H2AX and Rad51 foci at radiation induced DSBs.
(A) γ H2AX (red) and Rad51 (green) staining in E5 control retina. Nuclei were counterstained with DAPI (blue). Note the absence of any signals. (B-D) γ H2AX (red) and Rad51 (green) staining in E5 retina at 30 min after irradiation. Nuclei were counterstained with DAPI (blue). Note that γ H2AX foci appear in all nuclei whereas Rad51 foci are only visible in some nuclei of the retina and the RPE. (E) Quantification of γ H2AX foci in RPE cells devoid of Rad51 foci at 30 min after irradiation with 1 Gy (black, non-irradiated control). Data are presented as means (n = 3, with 10 nuclei analyzed for each experiment) ± SEM. (*** P<0.001). Scale bar = 10 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
Fig 2.
Lack of a G1/S checkpoint in retinal progenitor cells after DNA damage.
(A) Scheme of experimental design and quantification of BrdU+ cells in E5 embryos. BrdU was added at 3 hrs after 2 Gy irradiation. Fixation was done at 6 hrs after irradiation. (B) Staining against BrdU (green) in retinae of E5 controls and embryos irradiated with 2 Gy. Nuclei were counterstained with DAPI (blue). (C) Quantification of FACS cell cycle analysis from retinae of control and 2 Gy irradiated E5 embryos at 6 hrs after irradiation (n = 2). No differences in the ratio of S-phase cells were observed. (D) Scheme of experimental design and quantification of EdU+ and BrdU+ cells in E7 embryos. EdU was added directly after 2 Gy irradiation and BrdU was additionally applied at 3 hrs. Fixation was done at 6 hrs after irradiation. (E) Staining against EdU (red) and BrdU (green) in retinae of E7 controls and embryos irradiated with 2 Gy. Nuclei were counterstained with DAPI (blue). (F) Quantification of FACS cell cycle analysis of retinae from control and 2 Gy irradiated E7 embryos at 6 hrs after the treatment (n = 4). No differences in the ratio of S-phase cells were observed. Data are presented as means (n = 3, with sectors analyzed in central retinal regions that contain at least 400 cells for each experiment) ± SEM. Scale bar = 10 μm (B) and 20μm (E). RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
Fig 3.
X-ray-induced cell cycle arrest at G2/M-checkpoint in embryonic chick retina is abrogated at about 3 hours post-irradiation throughout development.
(A-F) Staining against the mitotic marker pH3 (red) in control (A-C) and 2 Gy (D-F) irradiated E3 retinae at 1, 3 and 6 hrs after irradiation. Nuclei were counterstained with DAPI (blue). Note absence of mitotic cells up to 3 hrs after irradiation. (G) Mitotic index in retinae of E3 embryos after irradiation with various doses at different time points. (H) Absolute numbers of mitotic events in retinal slices of E5 after irradiation with various doses at different time points. (I) Absolute numbers of mitotic events in retinal slices of E7 after irradiation with various doses at different time points. Note that at all stages, cells are released from G2-arrest around 3 hrs after irradiation. Data are presented as means (n = 3, with at least 100 cell analyzed for E3 and at least three different retinal slices analyzed for E5 and E7) ± SEM. (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
Fig 4.
Radiation-induced apoptotic events occur time- and dose-dependently between different stages of retinal development.
(A) cc3 staining (green) in control and in 2 Gy-irradiated E3 retinae at 12 hrs after irradiation. Nuclei were counterstained with DAPI (blue). (B) Representative DAPI staining of irradiated E3 retina including pyknotic nuclei (encircled in red) used for quantification of pyknosis. (C) Quantification of pyknotic nuclei at 12 hrs after irradiation with 0.5, 1, 2 and 4 Gy in central parts of E3 retinae. Note strong increase of apoptotic events after dose doubling from 0.5 to 1, but no further increase after doubling the dose from 1–2 Gy. (D) cc3 staining (green) in control and 2 Gy-irradiated E5 retinae at 12 hrs after irradiation. Nuclei were counterstained with DAPI (blue). (E) Quantification of pyknotic nuclei at 3–72 hrs after irradiation with 1 and 2 Gy in central parts of E5 retinae. Note the peak of radiation-induced apoptosis (RIA) at 12 hrs after irradiation and the linear correlation between doses and RIA. (F) cc3 staining (green) in control and 2 Gy-irradiated E7 retinae at 3 hrs after treatment. Nuclei were counterstained with DAPI (blue). (G) Quantification of pyknotic nuclei at 3–72 hrs after irradiation with 1 and 2 Gy in central parts of E7 retinae. Note the peak in RIA at 3 hrs after irradiation and absence of RIA after 1 Gy. Data are presented as means (n = 3 for E3, n = 7 for E5 and E7 with at least four different pictures analyzed for each experiment;) ± SEM (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm (A) and 50 μm (D, F). RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.
Fig 5.
Radiation-induced apoptotic events depend on basal-apical cell positions in E7 retina.
(A) For determination of the apical basal-to-apical positions of pyknotic nuclei within the retina, the tissue was subdivided in 5 bins ranging from the apical (No. 1) to the basal side (No. 5). (B) Quantification of pyknotic nuclei in E5 retinae 12 hrs after irradiation with 1 and 2 Gy. Note a slight shift in localization from apical to basal after doubling the dose from 1 to 2 Gy at 12 hrs. (C) Quantification of pyknotic nuclei in E7 retinae 3, 6 and 12 hrs after irradiation with 2 Gy. (D, E) Staining against cc3 (green) in E7 retinae at 3 and 12 hrs after irradiation with 2 Gy; nuclei were stained with DAPI (blue). Note a shift of apoptotic cells from basal to apical parts of the tissue from 6 to 12 hrs. Data are presented as means (n = 3, with at least four different pictures analyzed for each experiment); ± SEM (*P<0.05 **P< 0.01 *** P<0.001).