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Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation.

Mayer M, Kaiser N, Layer PG, Frohns F - PLoS ONE (2016)

Bottom Line: Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development.Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age.For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology and Neurogenetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0155093.g002: 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.

Mentions: Recent studies have shown the existence of a radiation-induced G2-arrest by the G2/M-checkpoint but no blockade of S-phase entry by a G1/S-checkpoint in the developing CNS of mice [3,4]. To analyze the impact of radiation on S-phase entry in the chick embryo, we first performed BrdU labeling studies. Since the G1/S-checkpoint has been described to be slowly activated at 4–6 hrs after irradiation of primary fibroblasts in vitro [15], BrdU was administered to E5 embryos at 3 hrs after the treatment. Fixation was performed at 6 hrs after irradiation (Fig 2A). Quantifications of BrdU-positive cells (BrdU+) in central parts of the retina revealed no significant changes (p-value = 0.844) between controls (49%) and 2 Gy irradiated samples (48%). (Fig 2A and 2B). FACS analysis of cell cycle distribution at 6 hrs after the irradiation confirmed the lack of changes in S-phase population but showed an increase in G2 phase cells after 2 Gy (Fig 2C, see S2A Fig for FACS blots).


Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation.

Mayer M, Kaiser N, Layer PG, Frohns F - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4862647&req=5

pone.0155093.g002: 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.
Mentions: Recent studies have shown the existence of a radiation-induced G2-arrest by the G2/M-checkpoint but no blockade of S-phase entry by a G1/S-checkpoint in the developing CNS of mice [3,4]. To analyze the impact of radiation on S-phase entry in the chick embryo, we first performed BrdU labeling studies. Since the G1/S-checkpoint has been described to be slowly activated at 4–6 hrs after irradiation of primary fibroblasts in vitro [15], BrdU was administered to E5 embryos at 3 hrs after the treatment. Fixation was performed at 6 hrs after irradiation (Fig 2A). Quantifications of BrdU-positive cells (BrdU+) in central parts of the retina revealed no significant changes (p-value = 0.844) between controls (49%) and 2 Gy irradiated samples (48%). (Fig 2A and 2B). FACS analysis of cell cycle distribution at 6 hrs after the irradiation confirmed the lack of changes in S-phase population but showed an increase in G2 phase cells after 2 Gy (Fig 2C, see S2A Fig for FACS blots).

Bottom Line: Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development.Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age.For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology and Neurogenetics, Darmstadt University of Technology, Darmstadt, Germany.

ABSTRACT
Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

No MeSH data available.


Related in: MedlinePlus