Ixed cultures was due to a change in transcript levels. Both

Ixed 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 UKI 1 web embryos (Figure S2 A and B). While this provides a simple explanation as to why there was no arterial reprogramming, analysis of tie1 tTA:tetOS nls-LacZ bigenic embryos at E10.5 (Figure 3F) and E13.5 (Figure 3G) exhibit positive b-gal staining within the dorsal aorta, suggesting that the absence of Prox1 in arterial endothelial cells is not due to an inefficiency of the bigenic system. Furthermore in Prox1 DT embryos, transcript expression from the driver construct was visualized via the VP16 antigen on both the dorsal aorta (arrowheads) and the jugular vein (arrows) (Figure 3H, Figure S3 and S4). The above observation therefore raises a fundamental question; when Prox1 is driven in both veins and arteries, how can arteries resist the forced expression of Prox1?DiscussionThe development of the mammalian vasculature is a highly organized and directed process, governed by genes that dictate the fate of endothelial cells to three major classes: venous, arterial and lymphatic. With the establishment of veins and arteries, the lymphatic vasculature is found to develop specifically from Calcitonin (salmon) venous and not arterial endothelial cells. One can envision a number of mechanisms that could restrict lymphangiogenesis to veins during embryonic development. For example, a unique molecular signature that defines venous endothelium may generate a specific signaling repertoire only accessible to Prox1; arterial endothelium having a different molecular profile would not support Prox1 mediated reprogramming to a lymphatic profile. Consistent with this hypothesis, venous and arterial endothelial cells have been found to display unique gene signatures [17,18]. Moreover, specific signaling pathways such as Notch, Sox18 and COUP-TFII play key roles in determining venous and arterial cell fate [4,16,22].Reprogramming via Prox1 in cultured venous and arterial endothelial cellsTo assess whether arterial endothelial cells (AECs) are amenable to reprogramming, AECs were engineered to overexpress Prox1 along with venous endothelial cells (VECs) as a control [21]. It was found that in culture, AECs and VECs engineered to overexpress Prox1 both underwent reprogramming that was consistent with its conversion to a lymphatic profile such as the downregulation of VEGFR-2, Tie2, Neuropilin-1 and STAT6, with the upregulation of VEGFR-3 and CyclinE2 (Figure 4A and B). This suggests that arterial endothelial cells can be molecularly reprogrammed to a lymphatic-like profile.Specificity of Vascular Reprogramming via ProxSpecificity of Vascular Reprogramming via ProxFigure 3. Reprogramming via Prox1 in double transgenics is restricted to veins. Immunohistochemistry on E13.5 controls and double transgenics stained with (A and C) Podoplanin or (B and D) LYVE-1. While the jugular veins of DT embryos stained positive for both markers (C and D, arrows), the dorsal aortas did not (arrowheads). (E) Furthermore, Prox1 expression is absent on the dorsal aorta (the DA identified using smooth muscle actin-FITC) in E13.5 double transgenics (arrowhead), in contrast to the clear presence of Prox1 (Cy3, arrows) on the jugular vein. The absence.Ixed 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 arterial reprogramming, analysis of tie1 tTA:tetOS nls-LacZ bigenic embryos at E10.5 (Figure 3F) and E13.5 (Figure 3G) exhibit positive b-gal staining within the dorsal aorta, suggesting that the absence of Prox1 in arterial endothelial cells is not due to an inefficiency of the bigenic system. Furthermore in Prox1 DT embryos, transcript expression from the driver construct was visualized via the VP16 antigen on both the dorsal aorta (arrowheads) and the jugular vein (arrows) (Figure 3H, Figure S3 and S4). The above observation therefore raises a fundamental question; when Prox1 is driven in both veins and arteries, how can arteries resist the forced expression of Prox1?DiscussionThe development of the mammalian vasculature is a highly organized and directed process, governed by genes that dictate the fate of endothelial cells to three major classes: venous, arterial and lymphatic. With the establishment of veins and arteries, the lymphatic vasculature is found to develop specifically from venous and not arterial endothelial cells. One can envision a number of mechanisms that could restrict lymphangiogenesis to veins during embryonic development. For example, a unique molecular signature that defines venous endothelium may generate a specific signaling repertoire only accessible to Prox1; arterial endothelium having a different molecular profile would not support Prox1 mediated reprogramming to a lymphatic profile. Consistent with this hypothesis, venous and arterial endothelial cells have been found to display unique gene signatures [17,18]. Moreover, specific signaling pathways such as Notch, Sox18 and COUP-TFII play key roles in determining venous and arterial cell fate [4,16,22].Reprogramming via Prox1 in cultured venous and arterial endothelial cellsTo assess whether arterial endothelial cells (AECs) are amenable to reprogramming, AECs were engineered to overexpress Prox1 along with venous endothelial cells (VECs) as a control [21]. It was found that in culture, AECs and VECs engineered to overexpress Prox1 both underwent reprogramming that was consistent with its conversion to a lymphatic profile such as the downregulation of VEGFR-2, Tie2, Neuropilin-1 and STAT6, with the upregulation of VEGFR-3 and CyclinE2 (Figure 4A and B). This suggests that arterial endothelial cells can be molecularly reprogrammed to a lymphatic-like profile.Specificity of Vascular Reprogramming via ProxSpecificity of Vascular Reprogramming via ProxFigure 3. Reprogramming via Prox1 in double transgenics is restricted to veins. Immunohistochemistry on E13.5 controls and double transgenics stained with (A and C) Podoplanin or (B and D) LYVE-1. While the jugular veins of DT embryos stained positive for both markers (C and D, arrows), the dorsal aortas did not (arrowheads). (E) Furthermore, Prox1 expression is absent on the dorsal aorta (the DA identified using smooth muscle actin-FITC) in E13.5 double transgenics (arrowhead), in contrast to the clear presence of Prox1 (Cy3, arrows) on the jugular vein. The absence.