Nal.pone.0052197.gSpecificity of Vascular Reprogramming via ProxSmooth muscle cell conditioned media does not downregulate ectopic Prox1 in arterial endothelial cellsWith the driver being able to express within the dorsal aorta it is curious that there appears to be no expression of Prox1, Title Loaded From File suggesting 1326631 that a mechanism may exist that restricts Prox1 expression from this vessel. Whether the suppression of Prox1 is through an endothelial cell non-autonomous or cell-autonomous mechanism is unclear. One event during embryonic development involves the early association (E9.5) of smooth muscle cells (SMCs) with the dorsa aorta; the cardinal vein appears without support cells at the equivalent time point (Figure 4C). Given the above observations, Prox1 expression may be modulated by a non-autonomous, soluble ligand-dependent mechanism derived from associated smooth muscle cells of the developing aorta. To address this, conditioned media from smooth muscle cells were used to culture AECs overexpressing Prox1 (AEC/Prox1). After 24 hours in SMC conditioned media, Prox1 Title Loaded From File levels did not mimic the decrease observed in vivo. In fact, there was an increase in Prox1 levels after AECs were exposed to conditioned media (Figure 4D). This suggests that a different mechanism exists to regulate Prox1 expression during embryonic development.Figure 2. Overexpression of Prox1 results in the expression of lymphatic markers on the jugular vein. (A) Normally, the expression of Podoplanin (FITC) on the jugular vein is downregulated by E13.5 and upregulated in lymph sacs, along with Prox1 (Cy3). (B) Prox1 overexpression results in its’ expression on the jugular vein as well as the lymph sac. Furthermore, Podoplanin is now found expressed on the jugular vein (arrows). Note that the lymph sac has become significantly enlarged. Similarly, immunohistochemistry on (C) control and (D) double transgenic E13.5 embryos show an increase in staining of LYVE-1 (arrows) on the lymph sac and jugular vein. Scale bar = 25 mm. JV: jugular vein; LS: lymph sac. doi:10.1371/journal.pone.0052197.gCell-cell interactions influence Prox1 mediated reprogramming in vitroTo explain the incongruence between our in vivo model and the conditioned media experiment, the answer may not lie with a freely soluble ligand but a direct cell-cell interaction. Specifically, we speculate that the inability to detect Prox1 in the dorsal aortas of DT embryos may be via direct interactions between smooth muscle cells and the arterial endothelium. To address this possibility, a mixing experiment was devised where equal cell numbers of AEC/Prox1 and SMCs were co-cultured. Significantly, it was observed that Prox1 expression was suppressed greater than two-fold upon co-culturing suggesting that the suppression of Prox1 is an active process (Figure 5A and B). This decrease was not due to differences in EC numbers upon mixing; Prox1 levels were normalized to EC content using Dil-Ac-LDL (Figure 5C). We next addressed whether the decrease in Prox1 observed in our AEC/SMC mixed cultures was due to a change in transcript levels. Both endpoint RT-PCR and quantitative RT-PCR analysis did not show any difference between the controls and mixed cultures suggesting that in our model Prox1 appears to be regulated at the post-transcriptional level (Figure 5D and E).positive cells are clearly present in control embryos, and more so in DT embryos (Figure S2 A and B). While this provides a simple explanation as to why there was no.Nal.pone.0052197.gSpecificity of Vascular Reprogramming via ProxSmooth muscle cell conditioned media does not downregulate ectopic Prox1 in arterial endothelial cellsWith the driver being able to express within the dorsal aorta it is curious that there appears to be no expression of Prox1, suggesting 1326631 that a mechanism may exist that restricts Prox1 expression from this vessel. Whether the suppression of Prox1 is through an endothelial cell non-autonomous or cell-autonomous mechanism is unclear. One event during embryonic development involves the early association (E9.5) of smooth muscle cells (SMCs) with the dorsa aorta; the cardinal vein appears without support cells at the equivalent time point (Figure 4C). Given the above observations, Prox1 expression may be modulated by a non-autonomous, soluble ligand-dependent mechanism derived from associated smooth muscle cells of the developing aorta. To address this, conditioned media from smooth muscle cells were used to culture AECs overexpressing Prox1 (AEC/Prox1). After 24 hours in SMC conditioned media, Prox1 levels did not mimic the decrease observed in vivo. In fact, there was an increase in Prox1 levels after AECs were exposed to conditioned media (Figure 4D). This suggests that a different mechanism exists to regulate Prox1 expression during embryonic development.Figure 2. Overexpression of Prox1 results in the expression of lymphatic markers on the jugular vein. (A) Normally, the expression of Podoplanin (FITC) on the jugular vein is downregulated by E13.5 and upregulated in lymph sacs, along with Prox1 (Cy3). (B) Prox1 overexpression results in its’ expression on the jugular vein as well as the lymph sac. Furthermore, Podoplanin is now found expressed on the jugular vein (arrows). Note that the lymph sac has become significantly enlarged. Similarly, immunohistochemistry on (C) control and (D) double transgenic E13.5 embryos show an increase in staining of LYVE-1 (arrows) on the lymph sac and jugular vein. Scale bar = 25 mm. JV: jugular vein; LS: lymph sac. doi:10.1371/journal.pone.0052197.gCell-cell interactions influence Prox1 mediated reprogramming in vitroTo explain the incongruence between our in vivo model and the conditioned media experiment, the answer may not lie with a freely soluble ligand but a direct cell-cell interaction. Specifically, we speculate that the inability to detect Prox1 in the dorsal aortas of DT embryos may be via direct interactions between smooth muscle cells and the arterial endothelium. To address this possibility, a mixing experiment was devised where equal cell numbers of AEC/Prox1 and SMCs were co-cultured. Significantly, it was observed that Prox1 expression was suppressed greater than two-fold upon co-culturing suggesting that the suppression of Prox1 is an active process (Figure 5A and B). This decrease was not due to differences in EC numbers upon mixing; Prox1 levels were normalized to EC content using Dil-Ac-LDL (Figure 5C). We next addressed whether the decrease in Prox1 observed in our AEC/SMC mixed cultures was due to a change in transcript levels. Both endpoint RT-PCR and quantitative RT-PCR analysis did not show any difference between the controls and mixed cultures suggesting that in our model Prox1 appears to be regulated at the post-transcriptional level (Figure 5D and E).positive cells are clearly present in control embryos, and more so in DT embryos (Figure S2 A and B). While this provides a simple explanation as to why there was no.