Effects of Prox1 on the migration of ESC-derived ECs and HUVECs. Cell migration was measured by video time-lapse microscopy as described in Materials and Methods. (A) ECs derived from Tc-Prox1 ESCs were subjected to video microscopy for 24 h (Supplementary Videos 1 and 2). (B) HUVECs were infected with adenoviruses (Ad) encoding DNA-binding mutant (Mut) or wild-type (WT) Prox1 and subjected to videomicroscopy for 24 h (Supplementary Videos 3 and 4). Results are expressed as the integrated cell motility over 24 h. Each value represents the mean of 10 determinations; bars, SD.

Effect of Prox1 on the migration of HUVECs stimulated with VEGF-A and VEGF-C. Cell migration was measured by Boyden chamber assay as described in Materials and Methods. HUVECs were infected with adenoviruses (Ad) encoding DNA-binding mutant (Mut) or wild-type (WT) Prox1 in combination with those encoding wild-type (WT), dominant-negative mutant form (d.n. Mut) of VEGFR3, or Null (encoding no transcripts) and plated on Boyden chambers with indicated chemoattractants placed in lower wells. Ad-Null was used in order to infect HUVECs with the same quantities of adenoviruses for all of samples. Results are expressed as the ratio of number of migrated cells normalized to control (no attractant). Each value represents the mean of triplicate determinations; bars, SD.

Roles of endogenous Prox1 in HDLECs. (A) Expression of endogenous Prox1 (left; green) was examined by specific antibodies in HUVECs (top) and HDLECs (bottom), with counterstaining for nuclei with propidium iodide (PI, middle; red; and right, merge). Bars, 100 μm. (B–E) Effects of Prox1 knockdown on expression of GAPDH (B), Prox1 (C), VEGFR3 (D), and integrin α9 (E) were examined by quantitative real-time PCR analysis. A scrambled siRNA sequence (Ambion) was used as a negative control siRNA (siNTC). Each value represents the mean of triplicate determinations; bars, SD. (F) Effects of Prox1 knockdown on the chemotaxes of HDLECs toward VEGF-A (50 ng/ml) and VEGF-C (50 ng/ml). Cell migration was measured by Boyden chamber assay as described in Materials and Methods. HDLECs were transfected with scrambled siRNAs (siNTC) or Prox1 siRNAs and plated on Boyden chambers with indicated chemoattractants placed in the lower wells. Results are expressed as the ratio of number of migrated cells normalized to control (no attractant). Each value represents the mean of triplicate determinations; bars, SD.

Effect of Tc-regulated Prox1 expression on the morphology and sheet formation of ESC-derived ECs and HUVECs. (A) Flk1+ endothelial progenitor cells were sorted from the differentiated ESCs carrying a Tc-regulated transgene encoding FLAG-epitope-tagged mouse Prox1 (Tc-Prox1) or control transgene (Tc-Empty) and redifferentiated in the presence (+) or absence (−) of Tc to obtain PECAM-1–positive ECs (bottom, red) and smooth muscle α-actin (SMA)-positive mural cells (bottom, green). Expression of FLAG-Prox1 (top, blue) and the morpholoby and sheet formation of ECs (bottom, red) were examined. Scale bars, 100 μm. (B) Quantitation of colony formation, EC and mural cell production, and endothelial sheet formation. Flk1+ cells derived from Tc-Prox1 ESCs were cultured sparsely with 10% fetal calf serum in the absence or presence of Tc for 4 d and stained for PECAM1 (red) and SMA (green). Numbers of different types of colonies per well were counted to determine the effects of Prox1 on colony formation of Flk1+ cells. Four colony types were observed: pure ECs forming sheet structures (EC-sheet, red); pure scattered ECs (EC-scattered, pink); pure mural cells (MC, green); and mixed colonies consisting of endothelial and mural cells (Mix, yellow). Experiments were repeated at least three times with essentially the same results. Bars, 50 μm. (C) Morphology of HUVECs infected with adenoviruses encoding LacZ, DNA-binding mutant (Mut), or wild-type (WT) Prox1. Bars, 100 μm.

Roles of integrin α9 in the migration of HUVECs. (A and B) Expression of transcripts for integrin α9 was determined by quantitative real-time PCR analysis in ESC-derived ECs (A) and HUVECs (B). (C and D) HUVECs were infected with adenoviruses encoding DNA-binding mutant (Mut) or wild-type (WT) Prox1 and were subjected to videomicroscopy for 24 h in the presence of control IgG or anti-integrin α9 (int α9)-neutralizing antibodies. The final images of HUVECs (C) and integrated cell motility (D) are shown. Bars, 100 μm. Each value represents the mean of 10 determinations; bars, SD (E) Cell migration was measured by Boyden chamber assay. HUVECs were infected with adenoviruses (Ad) encoding DNA-binding mutant (Mut) or wild-type (WT) and plated on Boyden chambers in the presence of control IgG or anti-integrin α9 (int α9)-neutralizing antibodies with VEGF-C placed in lower wells. Results are expressed as the ratio of number of migrated cells normalized to the control (no attractant). Each value represents the mean of triplicate determinations; bars, SD.

Expression of BEC and LEC markers in Prox1-expressing LECs derived from mouse embryos. E14 mouse embryos were dissected, and embryonic liver and spleen were removed. Other tissues were dissociated and subject to FACS sorting with anti-CD45, LYVE1 (ALY7), CD31, and CD34 antibodies (see Materials and Methods). CD45−; CD31+; CD34+; LYVE1− BEC fractions and CD45−; CD31+; CD34−; LYVE1+ LEC fractions were analyzed for the expression of transcripts for LYVE1 (A), Prox1 (B), VEGFR3 (C), and integrin α9 (D) by quantitative real-time PCR analysis. Each value represents the mean of triplicate determinations; bars, SD.

Schematic representation of the roles of Prox1 during the differentiation of blood vessel ECs (BECs) to lymphatic endothelial progenitor cells (LEPCs). See text for details.