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.

Figure 1

Induction of apoptotic cell death in the developing embryo after exposure to ionizing radiation. (A–F) Embryos received the indicated dose of ¹³⁷Cs gamma radiation at the early gastrula (6 hpf) stage, were allowed to continue development until 24 hpf and were processed to detect apoptotic cells by the TUNEL assay. Anterior is at top left, posterior is toward the bottom and the dark sphere is the yolk cell. Note that low levels of apoptosis occur during normal embryonic development. Arrows in panel C denote marked increase in apoptosis in the hindbrain and anterior spinal cord. Gross malformations (absence of eye, shortening of tail) are seen at higher doses (panels E–F). Embryos shown are from a single experiment and are representative of results obtained in several additional experiments.

Figure 2

Primary sequence and comparative analysis of zebrafish Ku80 protein. (A) Overall domain organization of eukaryotic Ku subunits. Ku70 and Ku80 have identical folds throughout much of their length (61). A von Willebrand Factor type A domain (VWA) domain is present at the N-terminus. The Ku core region, composed of a central β-barrel and small adjacent ‘arm’ domain, is present in all known eukaryotic and prokaryotic Ku homologs and forms the sliding clamp that encircles the DNA duplex. Small C-terminal domains mediate subunit-specific functions. (B) Phylogenetic tree showing relationship of zebrafish Ku80 to Ku80 in other vertebrates. (C) Sequence alignment of Ku80 proteins. Amino acid sequence is shown in the single-letter code. Asterisks denote identity; dots denote similarity shared among five vertebrate Ku80 proteins. Shown are zebrafish (zfish), pufferfish (pfish), Xenopus, mouse and human.

Figure 3

Expression of zebrafish Ku80 mRNA during embryogenesis. (A) Semi-quantitative RT–PCR analysis of Ku80 mRNA levels at the indicated developmental stages. (B–D) Whole-mount in situ hybridization to detect spatial expression of Ku80 mRNA. Ku 80 mRNA is uniform among blastomeres at the 2-cell (B) and early gastrula (6 hpf, C) stages. (D) At the Prim-5 stage (24 hpf) Ku80 mRNA accumulates in the developing retina (white arrow), otic vesicle (asterisk) and pronephric ducts (open arrows). Transverse sections of the midbrain (E) and isthmus (F) of the embryo shown in (G). Ku80 mRNA is expressed in cells along the ventricular surface (open arrows) and in retinal ganglion cells (black arrows). (G) Flat mount of 24 hpf embryo showing Ku80 mRNA expression in cells lining the brain ventricles. Vertical lines denote level of sections shown in (E) and (F). (H) Flat mount of 24 hpf embryos stained for phopho-Histone H3 (PH3) to identify cells in the G2/M transition of mitosis. PH3 positive cells (labeled in red) are located along the ventricular surface of the brain, similar to the pattern of Ku80 mRNA expression (compare white boxes in panels G and H). (I) At 48 hpf, Ku80 mRNA accumulates in pharyngeal mesenchyme (open triangles) and posterior tectum (filled arrows). (J) At 72 hpf, Ku80 mRNA is also detected in the proctodeum (open arrow). (D, I, and J) Lateral views with anterior to the left and dorsal to the top; (G and H) dorsal views with anterior to left.

Figure 4

Ku80 is required for radioprotection during embryogenesis. Embryos were exposed to the indicated doses of radiation (cGy) at 6 hpf, allowed to continue development and processed at 24 hpf to detect apoptosis by the TUNEL assay. (A, E, I) Uninjected embryos. (B, F, J) Embryos microinjected at 1-cell stage with ∼5 ng Ku80 morpholino oligonucleotide (Ku80 MO). (C, G, K) Embryos microinjected with a combination of ∼5 ng Ku80 MO and 200 ng of in vitro-transcribed Ku80 mRNA, prepared as described in Materials and Methods. The Ku80 MO is targeted against a 5′-untranslated sequence that is not present in the in vitro-synthesized Ku80 mRNA used for phenotypic rescue (18). (D, H, L) Embryos microinjected with 5 ng Ku80 mutant morpholino oligonucleotide (mutMO). All panels show lateral views of the anterior portion of embryo at 24 hpf. The asterisk denotes the otic vesicle; the open arrow denotes the retina. All panels are from the same experiment.

Figure 5

p53-mediated apoptosis in radiosensitive Ku80 morphants. Experimental design as in Figure 4. (A, E, I) Uninjected embryos. (B, F, J) Embryos injected at the 1-cell stage with ∼5 ng p53 morpholino oligonucleotide (P53 MO). (C, G, K) Embryos injected at the 1-cell stage with ∼5 ng Ku80 MO. (D, H, L) Embryos injected at the 1-cell stage with 5 ng Ku80 MO and 5 ng p53 MO. Orientation of embryos and labeling as in Figure 4. All panels are from the same experiment.