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Fig. S9
KLF2 regulation by APLNR and endothelial cell polarization in klf2 mutants.
(a) qPCR analysis. Validation of efficacy of siRNA for APLNR in HUVECs (left). KLF2 expression in APLNR KD HUVECs transfected with si SC or si APLNR subjected to laminar flow at 12 dynes/cm2 for 18 hours (right). *** P<0.05. KLF2 expression is downregulated in HUVECs after APLNR knock-down. (b-d) in situ hybridization for klf2a expression in 48 hpf wild-type (b), aplnrb+/- (c) and aplnrb -/-(d) embryos. The numbers of embryos examined are indicated in the top right corner of the images. (e) Brightfield images of 72 hpf wild-type, klf2a -/- and klf2b -/- larvae. (f) Quantification of larvae showing pericardial edema at 72 hpf. klf2a MO (2 and 4 ng) was injected into one-cell stage embryos from wild-type or klf2b+/- incrosses. (f-k′) Confocal images of 72 hpf Tg(kdrl:NLS-EGFP);Tg(fli1a:B4GALT1-mCherry) wild-type (g, g′), klf2a -/- (h, h′), klf2b-/- (i, i′), wild-type injected with klf2a MO (2ng) (j, j′) and klf2b-/- injected with klf2a MO (2ng) (k, k′) larvae. The white boxes in the left panels (g, h, i, j and k) are enlarged in the right panels (g′, h′, i′, j′ and k′). White dashed lines denote the ventral boundary of the DA. White arrowheads point to polarized ECs, yellow arrowheads to non-polarized ECs. The numbers of larvae examined are indicated in the top right corner of the images (g, h, i, j, k). (l-o′) Confocal images of 84 hpf TgBAC(etv2:EGFP) wild type (l, l′), klf2a -/- (m, m′) and klf2a -/-; klf2b+/- (n, n′) larvae. The red boxes in the left panels (l, m and n) are enlarged in the right panels (l′, m′ and n′). Red bars represent the diameter of the wild type DA, yellow bars the diameter of the wild-type PCV. (o) Quantification of blood vessel diameter in 84 hpf wild-type, klf2a -/- and klf2a -/-; klf2b+/- larvae. The numbers of larvae examined are indicated in the top of the graph. (p) Schematic representation of blood flow velocity measurements. (q) Quantification of blood flow velocity in 84 hpf wild-type, klf2a -/- and klf2a -/-; klf2b+/- larvae. The numbers of larvae examined are indicated in the top of the graph. Anterior to the left, dorsal to the top. Scale bars, 60 µm (b-d), 100 µm (e), 20 µm (g-n). DA, dorsal aorta; PCV, posterior cardinal vein. Error bars, SEM.