Figure 2.
Figure 2. Adenosine functions through A2b to regulate HSPC formation. (A and B) In situ hybridization of A2b at 24 (A) and 32 hpf (B). A2b is expressed in the vasculature of the AGM (red arrowheads) and the CHT (blue arrowheads). Insets are enlarged images of the AGM. (C) qPCR analysis of A2b expression in flk1:GFP+ FACS-sorted cells compared with flk1:GFP− cells at 23, 28, and 32 hpf. Results are shown as fold change of GFP+ to GFP− cells and normalized to expression of β-actin mRNA (Student’s t test: *, P < 0.05; n = 3 samples). (D) qPCR analysis of A2b expression in flk1:GFP+ cells from control or sih morphant embryos at the indicated stages: 16 and 23 hpf (before circulation) and 28 hpf (after circulation starts). Results are shown as fold change relative to A2b level at 16 hpf and normalized to expression of β-actin mRNA (Student’s t test: *, P < 0.05; n = 3 samples). (E) RT-PCR on uninjected or A2b splicing MO (A2b SP MO, here and following)–injected embryos with A2b- or actin-specific primers. Note the absence of correctly spliced A2b transcript after MO injection. (F–I) Expression of runx1/cmyb at 36 hpf in the AGM of control embryos (F), embryos injected with A2b MO (G), and hA2b mRNA–injected (H) and A2b MO- and hA2b mRNA–co-injected embryos (I). The numbers in each panel are combined from multiple experiments. (J and K) Expression of runx1/cmyb at 36 hpf in control embryos (J) and embryos treated with 10 µM BAY 60-6583 (K). (L–O) Expression of runx1/cmyb at 36 hpf in the AGM of control embryos (L) and A2b SP MO–injected (M), zA2b mRNA–injected (N), and A2b SP MO– and zA2b mRNA–co-injected embryos (O). (P–R′) Confocal images of Tg(cmyb:GFP; flk1:mcherry) embryos at 36 hpf. Control embryos (P and P′), embryos injected with A2b MO (Q and Q′), and embryos treated with 10 µM BAY 60-6583 (R and R′) are shown. White arrows mark the double-positive HSPCs. Blue arrowheads point to the pronephros. White dashed lines mark somite boundaries. (S) Quantification of the experiments in G and I. The results are presented as the mean percentage of embryos with restored runx1/cmyb staining as in I ± SE (Student’s t test: *, P < 0.05; n = 3 independent experiments, 20–25 embryos per experiment). (T) Quantification of number of HSPCs per somite in Tg(cmyb:GFP; flk1:mcherry) embryos. The results are presented as mean ± SE (Student’s t test: *, P < 0.05; **, P < 0.01; n = 5–8 embryos per group). Bars, 100 µm.

Adenosine functions through A2b to regulate HSPC formation. (A and B) In situ hybridization of A2b at 24 (A) and 32 hpf (B). A2b is expressed in the vasculature of the AGM (red arrowheads) and the CHT (blue arrowheads). Insets are enlarged images of the AGM. (C) qPCR analysis of A2b expression in flk1:GFP+ FACS-sorted cells compared with flk1:GFP− cells at 23, 28, and 32 hpf. Results are shown as fold change of GFP+ to GFP− cells and normalized to expression of β-actin mRNA (Student’s t test: *, P < 0.05; n = 3 samples). (D) qPCR analysis of A2b expression in flk1:GFP+ cells from control or sih morphant embryos at the indicated stages: 16 and 23 hpf (before circulation) and 28 hpf (after circulation starts). Results are shown as fold change relative to A2b level at 16 hpf and normalized to expression of β-actin mRNA (Student’s t test: *, P < 0.05; n = 3 samples). (E) RT-PCR on uninjected or A2b splicing MO (A2b SP MO, here and following)–injected embryos with A2b- or actin-specific primers. Note the absence of correctly spliced A2b transcript after MO injection. (F–I) Expression of runx1/cmyb at 36 hpf in the AGM of control embryos (F), embryos injected with A2b MO (G), and hA2b mRNA–injected (H) and A2b MO- and hA2b mRNA–co-injected embryos (I). The numbers in each panel are combined from multiple experiments. (J and K) Expression of runx1/cmyb at 36 hpf in control embryos (J) and embryos treated with 10 µM BAY 60-6583 (K). (L–O) Expression of runx1/cmyb at 36 hpf in the AGM of control embryos (L) and A2b SP MO–injected (M), zA2b mRNA–injected (N), and A2b SP MO– and zA2b mRNA–co-injected embryos (O). (P–R′) Confocal images of Tg(cmyb:GFP; flk1:mcherry) embryos at 36 hpf. Control embryos (P and P′), embryos injected with A2b MO (Q and Q′), and embryos treated with 10 µM BAY 60-6583 (R and R′) are shown. White arrows mark the double-positive HSPCs. Blue arrowheads point to the pronephros. White dashed lines mark somite boundaries. (S) Quantification of the experiments in G and I. The results are presented as the mean percentage of embryos with restored runx1/cmyb staining as in I ± SE (Student’s t test: *, P < 0.05; n = 3 independent experiments, 20–25 embryos per experiment). (T) Quantification of number of HSPCs per somite in Tg(cmyb:GFP; flk1:mcherry) embryos. The results are presented as mean ± SE (Student’s t test: *, P < 0.05; **, P < 0.01; n = 5–8 embryos per group). Bars, 100 µm.

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