Fig. 2

chk1 Knockdown Radiosensitizes p53 Mutants but Is Otherwise Compatible with Normal Zebrafish Development

(A) Fluorescent images of representative embryos of indicated genotypes +/- chk1 MO after 0 or 12.5 Gy IR; 5bpmmMO (5 base pair mismatch MO).

(B) Quantified AO responses of indicated genotypes with or without IR (12.5 Gy) and chk1 MO. Gray bars, p53^+/+ background; black bars, p53^e7/e7 background. AO staining was quantified in ≥8 embryos per condition, with > 1000 embryos scored. All data are reported as means ± SEM ^*** p < 0.0001 (two-tailed Student's t test).

(C) Western blots comparing the levels of Chk1, Chk2, and phosphorylated Cdc2 (Tyr15) in protein lysates from 25.5 hpf embryos injected with the indicated MOs.

(D) Nonirradiated p53^+/+;chk1^MO larva photographed live at 5 days postfertilization (dpf) show no apparent developmental defects but is slightly delayed (smaller swim bladder). Such larvae survived to adulthood.

(E) Fluorescent images of representative irradiated embryos of indicated genotypes. p53^e6 is the N168K mutation, corresponding to human residue 200. p53MO, MO against the p53 5′UTR.

(F) Fluorescent images of live transgenic embryos injected with the indicated MOs at the 1-cell stage and expressing EGFP in the notochord (top row, embryos photographed at 24 hpf) or in myeloid progenitors (bottom row, embryos photographed at 16.5 hpf). Tg(myoD:EGFP) and Tg(pu.1:EGFP) embryos were treated with or without IR (12.5 Gy) at 18 hpf and 10 hpf, respectively. Insets, higher magnification views of GFP-expressing cells. Top row, lateral views, anterior to the left. Bottom row, dorsal views, anterior facing down.

(G) Quantification of myeloid cells in 28 hpf embryos generated as indicated (x axis) and processed as in (H). Gray bars, p53^+/+ background; black bars, p53^e7/e7 background. mpo/l-plastin staining was quantified in ≥15 embryos per condition. Data are reported as means ± SD ^** p < 0.001, ^*** p < 0.0001 (two-tailed Student's t test). Note that while the numbers of mpo/l-plastin-positive cells are reduced ∼3-fold in IR-treated versus untreated p53^+/+ embryos; they are unchanged in treated versus untreated p53^e7/e7 embryos. Also note that chk1 knockdown induces an average 2-fold reduction in myeloid cell numbers in the p53^e7/e7 background after IR.

(H) Images of representative 28 hpf embryos of indicated genotypes processed for in situ hybridization of mpo and l-plastin riboprobes (blue, differentiated granulocytes and monocytes) and band 3 (red, erythrocytes). Note the specific reduction in number of granulocytes/monocytes.