(A) Sorted cell qPCR at 30 and 48 hpf for ezh1 and ezh2 expression on bulk Flk1−/Flt1− non-endothelial cells (NEC), Flk1+/Flt1− non-arterial endothelial cells (NAEC), Flk1+/Flt1+ arterial endothelium (AE), Gata2b+ hemogenic endothelial cells (HE), and Flk1+/cMyb+ hematopoietic stem and progenitor cells (HSPC). Reference gene: 18s (n ≥ 20 embryos/sample × ≥3 replicate clutches; two-way ANOVA, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Mean ± SEM). (B) Whole-embryo ezh2 qPCR on control and ezh1 morphants at 24 and 36 hpf relative to 18s (n ≥ 25 embryos/sample × 3 replicate clutches; two-tailed unpaired Student's t test, ∗p < 0.05. Mean ± SEM). (C) In vivo imaging of CD41:GFP+ cells in the CHT in control and ezh1 morphants ± dimethyl sulfoxide (DMSO)/1 μM GSK126 treatment from 12 to 72 hpf. Scale bar, 200 μm. (D) Quantification of fluorescent images in (C) (n = 7 embryos/condition; one-way ANOVA, ∗p < 0.05, ∗∗∗∗p < 0.0001. Error bars indicate SD). (E) Whole-embryo ephrinb2a, dll4, dlc, and tbx20 qPCR on DMSO and 1 μM GSK126-treated embryos from 12 to 24 hpf relative to 18s (n ≥ 25 embryos/sample × ≥3 replicate clutches; two-tailed unpaired Student's t test, ∗∗∗∗p < 0.0001. Mean ± SEM). (F) Whole-embryo ephrinb2a, dll4, dlc, and tbx20 qPCR on control and ezh2 mRNA-injected embryos at 24 hpf relative to 18s (n ≥ 25 embryos/sample × 3 replicate clutches; two-tailed unpaired Student's t test, not significant. Mean ± SEM). (G) WISH for gata2b on DMSO and 1 μM GSK126-treated embryos from 12 to 30 hpf. Scale bar, 100 μm. (H) Qualitative phenotypic distribution plot of embryos in (G) (n = 33 DMSO, 38 GSK126-treated embryos). (I) Whole-embryo gata2b qPCR on DMSO and 1 μM GSK126-treated embryos from 12 to 24 hpf relative to 18s (n ≥ 25 embryos/sample × 4 replicate clutches; two-tailed unpaired Student's t test, not significant. Mean ± SEM).
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