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

Abstract

The Ku70 protein, a product of the XRCC6 gene, is a component of the nonhomologous end-joining (NHEJ) pathway of DNA repair, which protects cells from the effects of radiation-induced DNA damage. Although the spatial expression of Ku70 during vertebrate embryogenesis has not been described, DNA repair proteins are generally considered to be "housekeeping" genes, which are required for radioprotection in all cells. Here, we report the cloning and characterization of the zebrafish Ku70 ortholog. In situ hybridization and RT-PCR analyses demonstrate that Ku70 mRNA is maternally provided and expressed uniformly among embryonic blastomeres. Later during embryogenesis, zygotically transcribed Ku70 mRNA specifically accumulates in neural tissue, including the retina and proliferative regions of the developing brain. In the absence of genotoxic stress, morpholino-mediated knockdown of Ku70 expression does not affect zebrafish embryogenesis. However, exposure of Ku70 morpholino-injected embryos to low doses of ionizing radiation leads to marked cell death throughout the developing brain, spinal cord, and tail. These results suggest that Ku70 protein plays a crucial role in protecting the developing nervous system from radiation-induced DNA damage during embryogenesis.

Figure 1

(A) Predicted amino acid sequence of zebrafish Ku70 aligned with that of human, mouse, and Xenopus. Identical residues in all sequences are marked with an “*”, conserved substitutions with a “:”, and semi-conservative substitutions with a “;”. Color coding of specific domains is according to the schematic in Panel B. (B) Cartoon representation of Ku70 peptide showing the domain structure and organization of zebrafish Ku70. Similar to other Ku70 proteins, zebrafish Ku70 has an N-terminal von Willebrand Factor type A domain (VWA), a central Ku core region, a Ku C-terminal arm, and SAP domain. The SAP domain (named after SAF-A/B, Acinus and PIAS motifs) is found in diverse nuclear proteins. The amino acid similarity between specific domains of zebrafish Ku70 and human Ku70 are shown below the structure, with amino acid identity in parentheses.

Figure 2

Expression of Ku70 mRNA during embryogenesis. (A) Temporal expression of Ku70 mRNA by RT-PCR analysis. Stages are as indicated; ss, somite stage; hpf, hours post-fertilization, M, DNA size marker. (B) Genomic structure of exons 1-3 and the locations of morpholino oligonucleotides. Electropherograms to the right show the wild-type exon1/exon2 boundary and the aberrant splicing of exon 1 to exon 3 in _sd2MO-injected embryos. (C-H) Expression of zebrafish Ku70 by in situ hybridization. (C) 2-cell stage. (D) 50% epiboly (∼6 hours post-fertilization). (E) 12 somite stage (ss), white arrows indicate three longitudinal proneural domains in the neural keel; r, retina. (F) 24 hpf; H, hindbrain; MHB, midbrain hindbrain boundary; T, tectum; D, diencephalon. Inset shows Ku70 mRNA expression in the developing tail (open triangles). (G) 48 hpf and (H) 72 hpf; Line arrow marks the tectum, solid black arrow marks the diencephalon, and triangles indicate the hindbrain. The otic vesicle is marked by an asterisk. Panels C, D, animal pole is toward top. Panels E, G, inset to F, and H are lateral views with anterior to left and dorsal toward top. Panel F is dorsal view, anterior toward bottom.

Figure 3

Expression of Ku70 is required for radioprotection. Whole mount TUNEL assay of zebrafish embryos at 24 hpf. 1-cell stage embryos were microinjected with buffer alone (A, F), Ku70 _atgMO (B, G), Ku70 _atgmmMO (C, H), Ku70 _sd2MO (D, I), or Ku70 _sd2mmMO (E, J). At 6 hpf, half of the embryos from each injection group were irradiated with 50 centiGray (cGy) of ionizing radiation (panels F-J) and the remaining embryos served as non-irradiated controls (panels A-E). All embryos were then allowed to develop to 24 hpf, when they were fixed and processed for the TUNEL assay. All panels are lateral view, head is to the top and tail is toward bottom.