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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 19, 2004 as doi:10.1096/fj.03-0903fje. |
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,2
* Faculty of Pharmacy and
Dept. of Pharmacology, University of Toronto, Toronto, Canada
2Correspondence: Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario M5S 2S2, Canada. Email: pg.wells{at}utoronto.ca;
SPECIFIC AIMS
This study sought to determine whether mice deficient in the ataxia telangiectasia mutated (ATM) gene were at increased risk for birth defects initiated by a model teratogen, ionizing radiation (Fig. 1
), that enhances formation of reactive oxygen species (ROS). The high in vivo radiosensitivity to low-dose ionizing radiation we observed in Atm null mutants stands in contrast to several in vitro studies that found cells from Atm null mice to be radioresistant. To examine this paradox, we explored the mechanism of cell death in wild-type and Atm null animals at several time points after low-dose irradiation at E10.5.
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PRINCIPAL FINDINGS
1. Atm is a teratologic suppressor after low-dose radiation during organogenesis
The most significant finding of this study is that ATM serves a teratological suppressor function protecting the embryo from radiation embryopathies during organogenesis. Atm protected embryos from teratogenesis initiated by even mild DNA damage from low-dose radiation.
Evidence included comprehensive characterization of embryopathies and associated genotoxic stress response. Atm-null, heterozygous Atm-deficient, and wild-type embryos were irradiated in vivo with 0.5 Gy (low dose) at E10.5. Irradiated Atm null fetuses were severely runted, with a 55% incidence of kinked tails (P<0.001) (Fig. 2
a, c), whereas no runting or kinked tails were observed in Atm+/– or wild-type animals. Similarly, postpartum lethality was sixfold higher in irradiated Atm null fetuses relative to Atm+/– or wild-type littermates (P=0.001). Otherwise, structural development was overtly normal in irradiated Atm null mice.
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In light of high sensitivity of Atm null embryos to low-dose radiation, potential radiosensitivity of heterozygous Atm-deficient embryos was examined using a fourfold higher radiation dose (2 Gy) at E10.5. Atm+/– fetuses were more radiosensitive than their wild-type littermates, with a 1.8-fold higher incidence of postpartum lethality (P=0.009). Resorption incidence in Atm+/– dams irradiated with 2 Gy was 2.7-fold higher than in Atm+/– dams irradiated with 0.5 Gy (P<0.001) and fourfold higher than in unirradiated Atm+/– dams (P<0.001). Accordingly, the embryonic genotype of resorptions resulting from a 2 Gy exposure was analyzed. Atm+/– embryos were highly radiosensitive, with a resorption incidence ninefold higher than that in wild-type embryos (P=0.006). After an exposure to 2 Gy, resorption incidence in Atm null embryos was 100%, which was 25-fold higher than in +/+ embryos (P<0.001) and approximately threefold higher than in +/– embryos (P<0.001). These results show that teratological response of both Atm null and heterozygous Atm-deficient animals to irradiation during organogenesis is shifted substantially toward increased susceptibility in a gene dose-dependent fashion.
2. Early "radioresistance" of Atm null (–/–) neuroepithelial progenitors
To understand in greater detail the mechanism of runting and tail kink anomalies observed in irradiated Atm null mice, we examined the process of cell death during embryonic development. Irradiation of wild-type E10.5 embryos with 0.5 Gy resulted in robust TUNEL labeling by 6 h postirradiation, particularly within proliferating neuroepithelium of the CNS. In contrast, Atm–/– embryos exposed to low-dose radiation showed little increase in TUNEL labeling during this period. Analysis of cell nuclei in parasagittal CNS sections with DAPI and by electron microscopy (Fig. 3
) confirmed these striking differences in relative levels of cellular degeneration in wild-type and Atm–/– embryos. The vast majority of degenerating cells observed in wild-type and Atm null embryos after irradiation were TUNEL-positive. These in vivo results demonstrate that, at 6 h after gamma-irradiation, neuroepithelia from Atm null mice are resistant to apoptotic degeneration compared with their wild-type littermates.
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3. Radiation induces a delayed and hybrid form of cell death in Atm null mutants
Dramatic early resistance of Atm null mice to radiation-induced cell death at E10.5 would seem to be at odds with the extreme radiosensitivity of this genotype when subsequently examined in vivo at fetal stage on E18.5. To investigate this paradox, cell death was examined at 48 h postirradiation. The level of TUNEL-positive cells in irradiated wild-type embryos was indistinguishable from that observed in unirradiated controls. In contrast, TUNEL labeling was substantially elevated in Atm null littermates at 48 h postirradiation, suggesting that, while proliferating neuroepithelim in Atm null mice is acutely resistant to programmed cell death after gamma-irradiation, damage induced by this treatment ultimately results in development of progeny that undergo an alternative form of programmed cell death. In contrast to the early, uniform pattern of programmed cell death observed in wild-type mice 6 h after irradiation, neuroepithelia in Atm null mice frequently died at 48 h in small clusters or localized groups. Comparison of spatial distribution of BrdU-positive and TUNEL-positive cells within the subventricular zone of wild-type and Atm null mice indicated that the primary zone of degeneration resided within the proliferative layer.
The nature of neuronal cell death observed in Atm null mice was also examined by measuring levels of activated caspase-3. Activated caspase-3 was absent in the CNS of wild-type embryos, but was broadly distributed in Atm null embryos at 48 h after irradiation at E10.5.
Ultrastructural analysis of neuroepithelia in telencephalon from irradiated wild-type and Atm null embryos is shown in Fig. 3
. At 48 h after irradiation, despite a high incidence of caspase-3-positive and TUNEL-positive labeling within degenerating cells of the subventricular zone of Atm –/– mice, along with a classic pattern of chromatin condensation, these cells displayed morphologic features inconsistent with apoptosis (Fig. 3e
). These inconsistencies included vacuolation, a characteristic of apoptosis that was particularly prominent during the latter phase of degeneration (Fig. 3i, j
). Ultimately, this resulted in extensive physical disruption of cell membrane and cell autolysis prior to formation of true apoptotic bodies (Fig. 3 k-m
).
In contrast to wild-type littermates, Atm null embryos at 6 h postirradiation do not respond with an increase in cell death. These data thus provide insights into the mechanism of Atm-dependent cell death in the developing nervous system at a gestational stage when Atm is highly expressed within the proliferating CNS. Neuroepithelia in Atm null mice appear resistant to acute apoptotic effects of gamma-irradiation, resulting in proliferation of irradiated neuroepithelia. In contrast to wild-type animals, where damaged cells normally are either eliminated by programmed cell death, or repair minor DNA damage and replicate normally, DNA-damaged cells in Atm null mice appear to replicate and produce progeny that subsequently undergo an aberrant form of programmed cell death due to loss of Atm.
CONCLUSIONS AND SIGNIFICANCE
In this in vivo study, we used a low dose (0.5 Gy) of ionizing radiation during organogenesis and demonstrated that Atm null embryos are exquisitely susceptible to radiation-initiated teratogenesis. Moreover, we observed that Atm null embryos respond to low-dose ionizing radiation with a hybrid form of delayed cell death that has characteristics of both apoptosis and necrosis.
The most significant finding of this research is that ATM serves a teratological suppressor function protecting the embryo from radiation embryopathies during organogenesis. Evidence includes a comprehensive characterization of embryopathies and the associated genotoxic stress response, and provides evidence for a hybrid form of cell death of neural progenitor cells in the embryonic central nervous system after exposure to well-characterized stimulus of ionizing radiation. This type of embryonic cellular response has not been observed previously, and reveals an alternative pathway by which the developing nervous system can respond to DNA damage. Our elucidation of this novel in vivo phenomenon resolves a previous paradox in the literature based upon in vitro studies, and contradicts published predictions of appropriate therapeutic strategies for patients with ATM deficiencies.
The enhanced susceptibility we observed in Atm heterozygotes is particularly important since heterozygosity for ATM mutations occur with an approximate frequency of 1 in 70 in humans. The exceptionally high susceptibility of Atm null mice to birth defects caused by even low-dose radiation observed herein is consistent with our previous discovery of a similar teratological suppressor role for p53, a downstream protein transduced by ATM and involved indirectly and directly in DNA repair, which protects the embryo from drugs that initiate formation of DNA-damaging ROS during organogenesis. We recently have obtained complementary preliminary evidence in embryo culture indicating that ATM also protects the embryo from both endogenous and drug-enhanced ROS, so the developmental role of ATM revealed in this radiation study likely has broader relevance to embryonic oxidative stress of any origin.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0903fje; 
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