Figure 1.
Figure 1. Increased hematopoietic cluster emergence in Hes-deficient embryonic aortas. (A) Transversal sections of embryos at 10.5 d showing expression of Hes5 and Hes1 by in situ hybridization (ISH). Arrowheads indicate the structures (Ao, aorta; IAHC, intra-aortic hematopoietic cluster; m, mesonephros; nt, neural tube; p2, p2 neural progenitors; SG, sympathetic ganglia). Details of IAHC and aorta in the right panels correspond to the boxed areas in the middle panels. (B) Relative expression of the indicated genes from E10.5 dissected AGMs after 3 d of 4-OH-tamoxifen treatments in explant cultures by qRT-PCR. Fold increase to the wild-type control after β2-Microglobulin and GAPDH normalization. Bars represent the mean ± SD determination from two experiments showing the fold increase versus the wild-type AGM. (C) Relative expression of Hes5 from sorted populations (endothelial, CD31−C-Kit−CD45−; hematopoietic clusters, CD31+C-Kit+) by qRT-PCR. mRNA was extracted from a pool of 10 AGMs at E10.5 in Hes1+/+ Hes5+/+ determinations and 4 AGMs in Hes1f/f Hes5+/− β-ACTIN–CRE-ERT after in vivo tamoxifen induction at E8. Bars represent the mean ± SD determination from one representative of two experiments showing the fold increase versus the wild-type AGM after β2-Microglobulin normalization. (D) Representative immunostaining of CD44 and SMA (arterial markers) in the AGM region in wild-type or Hes mutant embryos on E10.5 (A, dorsal aorta; Ao, aorta in AGM region; cv, cardinal vein; m, mesonephros; nt, neural tube). (E) Relative expression of endothelial (PECAM1), arterial markers (EphrinB2, smooth muscle actin [SMA], and CD44), and vein marker (EphB4) in E10.5 AGM after 3 d of 4-OH-tamoxifen treatment in explant culture from embryos with the indicated genotypes (n = 3 each genotype) by qRT-PCR. Bars represent the mean ± SD determination from three experiments showing the fold increase relative to the wild-type AGM. (F) Representative immunostaining of CD31 (endothelial and hematopoietic) and CD41 (hematopoietic precursors) in the AGM region in wild-type or Hes mutant embryos. Arrowheads indicate the structures. Bars: (A and D) 200 µm; (F) 50 µm. (G) Quantification of CD41+ cells per micrometer of AGM. Spearman test was used to assess the significance between wild-type allele number and CD41+ cells per micrometer. Correlation between number of wild-type alleles and CD41+ cells per micrometer was significant (**, P < 0.001; and ρ = 0.985). Bars represent the mean of counted cells in two different embryos ± SEM. (H) CD41+ clusters containing more than four cells per micrometer of AGM. Quantification of CD41+ cells per micrometer of AGM. Spearman correlation was used to assess the significance between number of wild-type alleles and CD41+ clusters containing more than four cells per micrometer (**, P = 0.005; and ρ = 0.803). Bars represent the mean of counted clusters in two different embryos ± SEM.

Increased hematopoietic cluster emergence in Hes-deficient embryonic aortas. (A) Transversal sections of embryos at 10.5 d showing expression of Hes5 and Hes1 by in situ hybridization (ISH). Arrowheads indicate the structures (Ao, aorta; IAHC, intra-aortic hematopoietic cluster; m, mesonephros; nt, neural tube; p2, p2 neural progenitors; SG, sympathetic ganglia). Details of IAHC and aorta in the right panels correspond to the boxed areas in the middle panels. (B) Relative expression of the indicated genes from E10.5 dissected AGMs after 3 d of 4-OH-tamoxifen treatments in explant cultures by qRT-PCR. Fold increase to the wild-type control after β2-Microglobulin and GAPDH normalization. Bars represent the mean ± SD determination from two experiments showing the fold increase versus the wild-type AGM. (C) Relative expression of Hes5 from sorted populations (endothelial, CD31−C-Kit−CD45−; hematopoietic clusters, CD31+C-Kit+) by qRT-PCR. mRNA was extracted from a pool of 10 AGMs at E10.5 in Hes1+/+ Hes5+/+ determinations and 4 AGMs in Hes1f/f Hes5+/− β-ACTIN–CRE-ERT after in vivo tamoxifen induction at E8. Bars represent the mean ± SD determination from one representative of two experiments showing the fold increase versus the wild-type AGM after β2-Microglobulin normalization. (D) Representative immunostaining of CD44 and SMA (arterial markers) in the AGM region in wild-type or Hes mutant embryos on E10.5 (A, dorsal aorta; Ao, aorta in AGM region; cv, cardinal vein; m, mesonephros; nt, neural tube). (E) Relative expression of endothelial (PECAM1), arterial markers (EphrinB2, smooth muscle actin [SMA], and CD44), and vein marker (EphB4) in E10.5 AGM after 3 d of 4-OH-tamoxifen treatment in explant culture from embryos with the indicated genotypes (n = 3 each genotype) by qRT-PCR. Bars represent the mean ± SD determination from three experiments showing the fold increase relative to the wild-type AGM. (F) Representative immunostaining of CD31 (endothelial and hematopoietic) and CD41 (hematopoietic precursors) in the AGM region in wild-type or Hes mutant embryos. Arrowheads indicate the structures. Bars: (A and D) 200 µm; (F) 50 µm. (G) Quantification of CD41+ cells per micrometer of AGM. Spearman test was used to assess the significance between wild-type allele number and CD41+ cells per micrometer. Correlation between number of wild-type alleles and CD41+ cells per micrometer was significant (**, P < 0.001; and ρ = 0.985). Bars represent the mean of counted cells in two different embryos ± SEM. (H) CD41+ clusters containing more than four cells per micrometer of AGM. Quantification of CD41+ cells per micrometer of AGM. Spearman correlation was used to assess the significance between number of wild-type alleles and CD41+ clusters containing more than four cells per micrometer (**, P = 0.005; and ρ = 0.803). Bars represent the mean of counted clusters in two different embryos ± SEM.

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