Figure 1
Figure 1. Fev is required for HSC development. (A) Whole-mount in situ hybridization shows expression of fev in lateral plate mesoderm (arrows) at 5-somite stage and in pancreas (red arrow) and blood vessels (black arrow) at 24 hpf. The right panel shows a cross-section of 24 hpf embryo in the trunk region at the location marked by the dotted line, showing fev expression in vessels. (B) Validation of fevMO (3 ng/embryo) by western blot in control and fev morphant embryos at 24 hpf. (C) The runx1 data, expression of runx1 at 24 and 36 hpf in embryos injected with control MO or fevMO (top panels). Arrows mark runx1 expression in HSCs. The cmyb data, expression of cmyb at 36 and 48 hpf in CHT of embryos injected with control MO (middle panels). The rag1 data, expression of rag1 at 4 dpf in thymus of embryos injected with control MO or fevMO (bottom panels). Arrows indicate CHT or thymus. Anterior is to the left and dorsal is up. (D) qPCR analysis of runx1 and cmyb expression in the dissected trunk region of embryos injected with control or fevMO at 24 and 36 hpf (mean ± SD, n = 3,*P < .05). (E) Population of the GFP+ cells (hematopoietic stem/progenitor cells) in the CHT region was reduced in cmyb:GFP/kdrl:mCherry double transgenic embryos injected with fevMO compared with those injected with control MO (top panels). White arrowheads mark HSCs/HSPCs. Population of the GFP+ cells in the pronephros and T cells in the thymus at 4 dpf were much reduced in cmyb:GFP/kdrl:mCherry double transgenic embryos injected with fevMO (bottom panels). The thymus and kidney are circled in yellow and white, respectively. (F) Quantification of GFP+ cells in the CHT, thymus, and kidney between control and fev morphant embryos in E. Note that 5 control embryos and 5 fev morphants were used for counting GFP+ cells in the CHT, thymus, and kidney regions. (G) Hemogenic endothelium analysis in the AGM region using a cmyb:GFP/kdr1:mCherry double transgenic line at 36 hpf. Blue arrow, kdrl+cmyb+ cells (hemogenic ECs); white arrowhead, kdrl−cmyb+ cells (emerging HSCs). CHT, caudal hematopoietic tissue.

Fev is required for HSC development. (A) Whole-mount in situ hybridization shows expression of fev in lateral plate mesoderm (arrows) at 5-somite stage and in pancreas (red arrow) and blood vessels (black arrow) at 24 hpf. The right panel shows a cross-section of 24 hpf embryo in the trunk region at the location marked by the dotted line, showing fev expression in vessels. (B) Validation of fevMO (3 ng/embryo) by western blot in control and fev morphant embryos at 24 hpf. (C) The runx1 data, expression of runx1 at 24 and 36 hpf in embryos injected with control MO or fevMO (top panels). Arrows mark runx1 expression in HSCs. The cmyb data, expression of cmyb at 36 and 48 hpf in CHT of embryos injected with control MO (middle panels). The rag1 data, expression of rag1 at 4 dpf in thymus of embryos injected with control MO or fevMO (bottom panels). Arrows indicate CHT or thymus. Anterior is to the left and dorsal is up. (D) qPCR analysis of runx1 and cmyb expression in the dissected trunk region of embryos injected with control or fevMO at 24 and 36 hpf (mean ± SD, n = 3,*P < .05). (E) Population of the GFP+ cells (hematopoietic stem/progenitor cells) in the CHT region was reduced in cmyb:GFP/kdrl:mCherry double transgenic embryos injected with fevMO compared with those injected with control MO (top panels). White arrowheads mark HSCs/HSPCs. Population of the GFP+ cells in the pronephros and T cells in the thymus at 4 dpf were much reduced in cmyb:GFP/kdrl:mCherry double transgenic embryos injected with fevMO (bottom panels). The thymus and kidney are circled in yellow and white, respectively. (F) Quantification of GFP+ cells in the CHT, thymus, and kidney between control and fev morphant embryos in E. Note that 5 control embryos and 5 fev morphants were used for counting GFP+ cells in the CHT, thymus, and kidney regions. (G) Hemogenic endothelium analysis in the AGM region using a cmyb:GFP/kdr1:mCherry double transgenic line at 36 hpf. Blue arrow, kdrl+cmyb+ cells (hemogenic ECs); white arrowhead, kdrl−cmyb+ cells (emerging HSCs). CHT, caudal hematopoietic tissue.

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