Figure 5.
Figure 5. klf9 mediates the TH/TR axis to regulate hematopoiesis. (A) The qRT-PCR results showed that the expression levels of klf9, thraa, and thrb were significantly upregulated in the T3-treated zebrafish embryos compared with the controls at 3 dpf (means ± SD; **P < .01; n = 3). (B) O-dianisidine (O-D) staining of hemoglobin and be2-globin expression by WISH revealed that mature erythrocytes were decreased in klf9 morphants compared with control embryos at 48 dpf. RBCs are indicated by arrowheads. Scale bar, 100 μm. (C) Confocal imaging of Tg(gata1:dsRed) showing fewer gata1:dsRed cells in klf9 morphants at 48 hpf, 3 dpf, and 4 dpf. Scale bar, 100 μm. (D) Blood smear and Giemsa staining. At 4 dpf, the size and nucleocytoplasmic ratio of RBCs from klf9 morphants were increased compared with those from controls. The arrowhead marks the mature RBCs. Scale bar, 10 μm. (E) Scatter plots of the nucleocytoplasmic ratio in control and klf9 morphants (means ± SD; **P < .01; n = 20). (F) The qRT-PCR result displayed that klf9 expression was increased in gata1+ cells after T3 treatment from 10 hpf to 3 dpf (means ± SD; **P < .01; n = 5). (G) The qRT-PCR results showed that ae1-globin and lck expression levels were significantly decreased in klf9 morphants compared with controls at 4 dpf (means ± SD; **P < .01; n = 3). (H) WISH result showed that the expression of T-cell marker rag1 was decreased in klf9 morphants at 4 dpf. The white dashed lines indicate the thymus region. Scale bars, 100 μm. (I) Confocal imaging of Tg(coro1a:GFP) showed that T cells in the thymus (dashed line) were decreased in klf9 morphants compared with controls at 5.5 dpf. Scale bars, 40 μm. (J-K) The quantification of apoptotic cells in the caudal hematopoietic tissue (white boxes) showed that the number of TUNEL+ cells (arrowhead) was 1.5-fold higher in morphants than that in controls at 2 dpf (means ± SD; *P < .05; Ctrl, n = 5; MO, n = 4). Scale bars, 100 μm. (L) The qRT-PCR results showed that the expression levels of cell division–associated genes were downregulated in klf9 morphants at 5 dpf (means ± SD; **P < .01; n = 3). Ctrl, control; MO, klf9 MO.

klf9 mediates the TH/TR axis to regulate hematopoiesis. (A) The qRT-PCR results showed that the expression levels of klf9, thraa, and thrb were significantly upregulated in the T3-treated zebrafish embryos compared with the controls at 3 dpf (means ± SD; **P < .01; n = 3). (B) O-dianisidine (O-D) staining of hemoglobin and be2-globin expression by WISH revealed that mature erythrocytes were decreased in klf9 morphants compared with control embryos at 48 dpf. RBCs are indicated by arrowheads. Scale bar, 100 μm. (C) Confocal imaging of Tg(gata1:dsRed) showing fewer gata1:dsRed cells in klf9 morphants at 48 hpf, 3 dpf, and 4 dpf. Scale bar, 100 μm. (D) Blood smear and Giemsa staining. At 4 dpf, the size and nucleocytoplasmic ratio of RBCs from klf9 morphants were increased compared with those from controls. The arrowhead marks the mature RBCs. Scale bar, 10 μm. (E) Scatter plots of the nucleocytoplasmic ratio in control and klf9 morphants (means ± SD; **P < .01; n = 20). (F) The qRT-PCR result displayed that klf9 expression was increased in gata1+ cells after T3 treatment from 10 hpf to 3 dpf (means ± SD; **P < .01; n = 5). (G) The qRT-PCR results showed that ae1-globin and lck expression levels were significantly decreased in klf9 morphants compared with controls at 4 dpf (means ± SD; **P < .01; n = 3). (H) WISH result showed that the expression of T-cell marker rag1 was decreased in klf9 morphants at 4 dpf. The white dashed lines indicate the thymus region. Scale bars, 100 μm. (I) Confocal imaging of Tg(coro1a:GFP) showed that T cells in the thymus (dashed line) were decreased in klf9 morphants compared with controls at 5.5 dpf. Scale bars, 40 μm. (J-K) The quantification of apoptotic cells in the caudal hematopoietic tissue (white boxes) showed that the number of TUNEL+ cells (arrowhead) was 1.5-fold higher in morphants than that in controls at 2 dpf (means ± SD; *P < .05; Ctrl, n = 5; MO, n = 4). Scale bars, 100 μm. (L) The qRT-PCR results showed that the expression levels of cell division–associated genes were downregulated in klf9 morphants at 5 dpf (means ± SD; **P < .01; n = 3). Ctrl, control; MO, klf9 MO.

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