(A) Induction of apoptosis in hematopoietic cells of the zebrafish embryo. Both a low magnification of the whole body and higher magnification of the trunk region are shown in (a) and (c), respectively. White squares in the low magnification images designate the regions shown in the higher magnification images in the adjoining right panels. (a) Apoptotic cells were detected by a TUNEL assay of the ventral wall of the dorsal aorta (VW-DA) (shown by green arrow heads in the right panel) at 28 hpf. (b) Quantification of the TUNEL-positive cells in the ventral wall of dorsal aorta (VW-DA) at 28 hpf. The number of TUNEL-positive cells was estimated within the gated area indicated by the green dashed rectangle at the upper yolk extension. (c) Apoptotic cells were detected by a TUNEL assay of the caudal venous plexus (CVP) (shown by green arrow heads in the right panel) at 48 hpf. (d) Quantification of the TUNEL-positive cells detected in the CVP at 48 hpf. The quantity of TUNEL-positive cells was assessed.within the gated area indicated by the green dashed rectangle at the anatomical CVP region. (e) Detection of apoptosis in gata-1^GFP-positive hematopoietic cells. Transverse sections through the trunk region of 48-hpf gata-1^GFP embryos with the dorsal up are shown. The caudal artery (CA; upper) and caudal vein (CV; lower) are shown by orange arrow heads in the panels. By TUNEL assay, gata-1^GFP-positive apoptotic cells in the CV are evident and indicated by white arrows. (B) Lateral views of 48, 72 and 120 hpf embryos following the co-injection of zTERT-MO1 (or Cont-MO1) and GFP-zTERT-cDNA (or GFP-cDNA) expression vectors (a–l); black arrowheads indicate the heart regions. Bright field pictures of blood cells in trunks of 72 hpf embryos after co-injection of zTERT-MO1 or Cont-MO1 and either a GFP-cDNA or GFP-zTERT-cDNA vector (m–p). The upper vessel is the dorsal artery (from left to right arrows) and the lower vessel is the posterior cardinal vein (from right to left arrows). (C) Quantitation of the circulating blood cell number in zTERT-MO- (blue circle) versus Cont-MO- (black open circle) injected embryos during 28–72 hpf (a). (b) Calculation of the percentage of the control circulating blood cell numbers at 72 hpf after co-injection of zTERT-MO or Cont-MO and either a GFP-control or GFP-zTERT-cDNA vector. Blood cell numbers were determined for 10 embryos from each group. (D) Whole-mount o-dianisidine staining for heme detection in uninjected, Cont-MO1- and zTERT-MO1-injected embryos during 32–168 hpf. Blood flow over the yolk sac and in the tail vessels results in brown staining in wild type (data not shown) and Cont-MO1-injected embryos during 32–168 hpf (ventral view). (E) H&E staining of blood cells in tissue sections of the arteries or veins of Cont-MO1- and TERT-MO1-injected embryos at 33 and 48 hpf, and Wright-Giemsa staining of isolated blood cells from Cont-MO1- and TERT-MO1-injected embryos at 48 hpf‌‌.

https://doi.org/10.1371/journal.pone.0350446.g002

(A) Lateral views (anterior to left) of wild-type (p53^+/+) and homozygous p53^M214K mutant (p53^m/m) embryos injected with TERT-MO1 (a–d). Arrowheads indicate the heart regions, including the blood (a’–d’) and views of the artery and veins (anterior to left) in the trunk at 72 hpf. (B) Scoring system based on the number of circulating blood cells at 72 hpf after injection of zTERT-MO1 or Cont-MO1 into p53^+/+ and p53^m/m embryos. We divided the embryos into three classes based on their flowing blood cell number: i) indistinguishable from the control (>90%; no change) as indicated by the gray bar, ii) cell number reduction compared with the control (10–90%; reduction) as indicated by the light-blue bar, and iii) severely deficient or almost no flowing blood cells (<10%; deficiency) as indicated by the dark-blue bar. (C) Percentages of the control levels of circulating blood cell numbers at 72 hpf after the injection of zTERT-MO1 or Cont-MO1 into p53^+/+ and p53^m/m embryos. Blood cell numbers were counted in 10 embryos from each group. ^*P < 0.01 (Student t-test). (D) Whole-mount TUNEL staining in control and zTERT-knockdown embryos coinjected with either zp53-MO- or zp53-control-MO at 48 hpf (a–d). A representative region of TUNEL-positive cells is indicated by the brackets (c, d). (E) Whole-mount o-dianisidine staining of hemoglobin in control and TERT-knockdown embryos coinjected with either zp53-MO or zp53-control-MO at 48 hpf. The intensity of the blood flow color over the yolk indicates the hemoglobin concentration (a–d). Wright-Giemsa staining of isolated blood cells from Cont-MO1- and zTERT-MO1-injected embryos in a p53-deficient background at 48 hpf (a’–d’). (F) Whole-mount in situ hybridization of control and TERT-knockdown embryos for c-myb and runx1 expression in p53^+/+ and p53^m/m embryos. The expression in the arterial region is indicated by arrowheads. (G) Altered expression levels of the indicated genes in TERT-deficient embryos in a p53-deficient background. Genes involved in the p53 pathway were analyzed by single-embryo RT-PCR. Similar results (data not shown) were obtained from this analysis of a number of individual embryos (more than 10 embryos for each gene). (H) Scoring of the number of circulating blood cells at 72 hpf after co-injection of zTERT-MO1 or Cont-MO1 and either GFP-, hBcl-2-, or E1B-19K-cDNA vectors. Embryos are classified as in (B). (I) Circulating blood cell numbers as a percentage of the control at 72 hpf after co-injection of zTERT-MO1 or Cont-MO1 and either GFP-control, hBcl-2, or E1B-19K expressing vectors. Blood cell numbers were counted in 10 embryos for each group. ^*P < 0.01 (Student t-test).

https://doi.org/10.1371/journal.pone.0350446.g005