Ally derived from veins and not arteries.the embryo and underscores the importance of the regulated expression of Prox1 in vascular development.Results Double transgenic embryos suffer from edemaThe early development of the lymphatic vasculature depends on the regulated expression of Prox1 on the cardinal vein. During this event, lymphatic precursor cells bud off from the vein and migrate outward in a directional fashion to form the primordial lymph sac [10,11]. Prox1 ablation results in the dedifferentiation of lymphatic endothelial cells to a more vascular cell-like identity, suggesting that this transcription factor is required for lymphatic differentiation [11,19]. To further extend these observations, we have generated a transgenic model where one can ML240 ectopically express Prox1 specifically in blood endothelial cells in order to demonstrate that Prox1 leads to the genetic reprogramming of the vasculature (Figure 1A) [15]. Indeed, in vitro data demonstrates that the overexpression of Prox1 generates a shift in the gene signature of vascular endothelial cells to a lymphatic cell profile [13,14]. Upon Prox1 overexpression in blood endothelial cells, late stage embryos display significant edema and anemia at E14.5 (Figure 1B and C). Previous results have demonstrated a distended lymph sac and separation of the epidermis from the dermis typical of a defect in lymphatic function [15]. Clearly, the overexpresion of Prox1 in blood endothelial cells has a negative effect on the development ofDifferences in the reprogramming of veins and arteries in DT embryosNext, we investigated whether reprogramming via Prox1 can be reproduced in vivo. Consistent with Schacht et al., in E13.5 control embryos Podoplanin expression becomes downregulated on the jugular vein with Prox1 expression being absent [20]. In contrast, Prox1 and Podoplanin are expressed on the jugular vein of double transgenic (DT) embryos (Figure 2A and B, arrows, Figure S1), along with LYVE-1 (Figure 2C and D, arrows). These results suggest that the blood vasculature is indeed malleable and that the overexpression of Prox1 can alter the profile of vascular endothelial cells to a more lymphatic phenotype in vivo. The above data points to the plasticity of the blood vascular system to Prox1 reprogramming, however an interesting exception was observed. Later in development, arterial endothelial cells in DT embryos appear resistant to reprogramming. At E13.5, markers such as Podoplanin (Figure 3A and C, arrowhead) and LYVE-1 (Figure 3B and D, arrowhead) are absent on the arteries of DT embryos. Upon further investigation, it was found that the arterial vessels of E13.5 DT embryos did not ectopically express Prox1, in contrast to the jugular vein and lymph sacs (Figure 3E, arrowhead, Figure S5). Indeed, by E11.5 Prox1 expression appears to be suppressed on the dorsal aortas of DT embryos. Of note, ProxFigure 1. Overexpression of Prox1 in the blood vasculature results in edema and embryonic CASIN biological activity lethality at E14.5. Gross analysis of embryos at E14.5 from control and double transgenic (DT) embryos for tie1 tTA:tetOS prox1. (A) Bigenic transgene construction. The absence of doxycycline is molecularly permissive for transgene expression. In contrast, the presence of doxycycline suppresses transgene expression. (B) Control embryos display typical architecture for blood vasculature, however transgenic overexpression of Prox1 results in edema, anemia and lethality. Scale bar = 1 cm. doi:10.1371/jour.Ally derived from veins and not arteries.the embryo and underscores the importance of the regulated expression of Prox1 in vascular development.Results Double transgenic embryos suffer from edemaThe early development of the lymphatic vasculature depends on the regulated expression of Prox1 on the cardinal vein. During this event, lymphatic precursor cells bud off from the vein and migrate outward in a directional fashion to form the primordial lymph sac [10,11]. Prox1 ablation results in the dedifferentiation of lymphatic endothelial cells to a more vascular cell-like identity, suggesting that this transcription factor is required for lymphatic differentiation [11,19]. To further extend these observations, we have generated a transgenic model where one can ectopically express Prox1 specifically in blood endothelial cells in order to demonstrate that Prox1 leads to the genetic reprogramming of the vasculature (Figure 1A) [15]. Indeed, in vitro data demonstrates that the overexpression of Prox1 generates a shift in the gene signature of vascular endothelial cells to a lymphatic cell profile [13,14]. Upon Prox1 overexpression in blood endothelial cells, late stage embryos display significant edema and anemia at E14.5 (Figure 1B and C). Previous results have demonstrated a distended lymph sac and separation of the epidermis from the dermis typical of a defect in lymphatic function [15]. Clearly, the overexpresion of Prox1 in blood endothelial cells has a negative effect on the development ofDifferences in the reprogramming of veins and arteries in DT embryosNext, we investigated whether reprogramming via Prox1 can be reproduced in vivo. Consistent with Schacht et al., in E13.5 control embryos Podoplanin expression becomes downregulated on the jugular vein with Prox1 expression being absent [20]. In contrast, Prox1 and Podoplanin are expressed on the jugular vein of double transgenic (DT) embryos (Figure 2A and B, arrows, Figure S1), along with LYVE-1 (Figure 2C and D, arrows). These results suggest that the blood vasculature is indeed malleable and that the overexpression of Prox1 can alter the profile of vascular endothelial cells to a more lymphatic phenotype in vivo. The above data points to the plasticity of the blood vascular system to Prox1 reprogramming, however an interesting exception was observed. Later in development, arterial endothelial cells in DT embryos appear resistant to reprogramming. At E13.5, markers such as Podoplanin (Figure 3A and C, arrowhead) and LYVE-1 (Figure 3B and D, arrowhead) are absent on the arteries of DT embryos. Upon further investigation, it was found that the arterial vessels of E13.5 DT embryos did not ectopically express Prox1, in contrast to the jugular vein and lymph sacs (Figure 3E, arrowhead, Figure S5). Indeed, by E11.5 Prox1 expression appears to be suppressed on the dorsal aortas of DT embryos. Of note, ProxFigure 1. Overexpression of Prox1 in the blood vasculature results in edema and embryonic lethality at E14.5. Gross analysis of embryos at E14.5 from control and double transgenic (DT) embryos for tie1 tTA:tetOS prox1. (A) Bigenic transgene construction. The absence of doxycycline is molecularly permissive for transgene expression. In contrast, the presence of doxycycline suppresses transgene expression. (B) Control embryos display typical architecture for blood vasculature, however transgenic overexpression of Prox1 results in edema, anemia and lethality. Scale bar = 1 cm. doi:10.1371/jour.