DNA damage response and Ku80 function in the vertebrate embryo

Abstract

Cellular responses to DNA damage reflect the dynamic integration of cell cycle control, cell–cell interactions and tissue-specific patterns of gene regulation that occurs in vivo but is not recapitulated in cell culture models. Here we describe use of the zebrafish embryo as a model system to identify determinants of the in vivo response to ionizing radiation-induced DNA damage. To demonstrate the utility of the model we cloned and characterized the embryonic function of the XRCC5 gene, which encodes Ku80, an essential component of the nonhomologous end joining pathway of DNA repair. After the onset of zygotic transcription, Ku80 mRNA accumulates in a tissue-specific pattern, which includes proliferative zones of the retina and central nervous system. In the absence of genotoxic stress, zebrafish embryos with reduced Ku80 function develop normally. However, low dose irradiation of these embryos during gastrulation leads to marked apoptosis throughout the developing central nervous system. Apoptosis is p53 dependent, indicating that it is a downstream consequence of unrepaired DNA damage. Results suggest that nonhomologous end joining components mediate DNA repair to promote survival of irradiated cells during embryogenesis.

Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

“…In part, this is due to the a priori assumption that many, if not most, of the DNA repair proteins are expressed ubiquitously. However, in contrast to this expectation, we have shown that expression of Ku80 mRNA varies greatly among tissues during embryogenesis, with expression highest in developing neural tissues [19]. …”

“…2H). The domains of zebrafish Ku70 mRNA expression are similar to those of Ku80 and suggest that, like Ku80, Ku70 is predominantly expressed in proliferative regions of the developing nervous system, such as the ventricular zones of the brain and presumptive ganglion cell layer of the retina [19].…”

“…2H). The domains of zebrafish Ku70 mRNA expression are similar to those of Ku80 and suggest that, like Ku80, Ku70 is predominantly expressed in proliferative regions of the developing nervous system, such as the ventricular zones of the brain and presumptive ganglion cell layer of the retina [19].To determine whether expression of Ku70 is required for neural development, we designed an antisense MO [26] complementary to the Ku70 translation initiation site ( atg MO; Fig. 2B) and a control MO containing a 5-base mismatch ( atgmm MO).…”

“…Dosimetry was performed using thermoluminescent dosimetry devices (Landauer Inc, Glenwood, IL) irradiated simultaneously with embryos. Irradiated embryos were fixed at 24 hpf and assayed by the TUNEL method [19].…”

Functional characterization of the Bcl-2 gene family in the zebrafish

“…Exposure to g-irradiation induced a substantial increase in activated caspase-3, which was dependent on p53 ( Figure 4a). 25 Ectopic expression of prosurvival Bcl-2 family members, such as zMcl-1a, zMcl-1b, zBlp1, or zBlp2, protected embryos from g-irradiation-induced apoptosis (Figure 4b). Thus, Bcl-2 family genes control activation of the intrinsic pathway in response to g-irradiation in zebrafish as well as in mammals.…”