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Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature.

Kim H, Nguyen VP, Petrova TV, Cruz M, Alitalo K, Dumont DJ - BMC Dev. Biol. (2010)

Bottom Line: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature.Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sunnybrook Research Institute University of Toronto 2075 Bayview Avenue Toronto, Ontario M4N 3M5, Canada. dan.dumont@sri.utoronto.ca

ABSTRACT

Background: In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo.

Results: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.

Conclusion: We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

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Analysis of reprogramming in yolk sacs from Prox1 double transgenic embryos. (A) Analysis of control and DT embryos at E10.5 (lanes 1 and 2) and yolk sacs at E10.5 or E12.5 (lanes 3 to 6) for prox1 expression by RT-PCR. (B) Yolk sacs from E13.5 control and DT embryos were analyzed using RT-PCR for the expression of lymphatic associated markers such as CyclinE2, Stat6, VEGFR-2, VEGFR-3 and Neuropilin-1 (NRP1). -ve represents no template control. (C) Similarly, yolk sacs from E12.5 control and DT embryos were analyzed by western blot for the expression of VEGFR-2, VEGFR-3, Tie2 and Neuropilin-2 (NRP2).
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Figure 3: Analysis of reprogramming in yolk sacs from Prox1 double transgenic embryos. (A) Analysis of control and DT embryos at E10.5 (lanes 1 and 2) and yolk sacs at E10.5 or E12.5 (lanes 3 to 6) for prox1 expression by RT-PCR. (B) Yolk sacs from E13.5 control and DT embryos were analyzed using RT-PCR for the expression of lymphatic associated markers such as CyclinE2, Stat6, VEGFR-2, VEGFR-3 and Neuropilin-1 (NRP1). -ve represents no template control. (C) Similarly, yolk sacs from E12.5 control and DT embryos were analyzed by western blot for the expression of VEGFR-2, VEGFR-3, Tie2 and Neuropilin-2 (NRP2).

Mentions: Given the influence of Prox1 on gene regulation in vascular and lymphatic endothelial cells, we further investigated the possibility of a switch in the molecular identity of endothelial cells using markers that are influenced by Prox1. To this end, we started our analysis with the yolk sac, a relatively simple tissue that is rich in blood vasculature (Figure 3a). Analysis of control or DT embryos reveal that the overexpression of Prox1 in vascular endothelial cells could alter gene expression to that of a more lymphatic endothelial cell signature. For example, transcripts for genes such as VEGFR-3 and CyclinE2 increase while VEGFR-2, Neuropilin1 and Stat6 decrease (Figure 3b). Furthermore, protein levels from yolk sacs derived from DT embryos demonstrate that VEGFR-2 and Tie2 decrease while Neuropilin-2 increases with a marginal increase in VEGFR-3. Wholemount analysis of DT yolk sacs support the reprogramming event, indicated with an increase in Podoplanin expression that correlates with Prox1 overexpression (data not shown). Consistent with previous findings, the overexpression of Prox1 in vascular endothelial cells appear to sufficiently alter the gene signature of BECs to that of a more LEC profile in vivo.


Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature.

Kim H, Nguyen VP, Petrova TV, Cruz M, Alitalo K, Dumont DJ - BMC Dev. Biol. (2010)

Analysis of reprogramming in yolk sacs from Prox1 double transgenic embryos. (A) Analysis of control and DT embryos at E10.5 (lanes 1 and 2) and yolk sacs at E10.5 or E12.5 (lanes 3 to 6) for prox1 expression by RT-PCR. (B) Yolk sacs from E13.5 control and DT embryos were analyzed using RT-PCR for the expression of lymphatic associated markers such as CyclinE2, Stat6, VEGFR-2, VEGFR-3 and Neuropilin-1 (NRP1). -ve represents no template control. (C) Similarly, yolk sacs from E12.5 control and DT embryos were analyzed by western blot for the expression of VEGFR-2, VEGFR-3, Tie2 and Neuropilin-2 (NRP2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909156&req=5

Figure 3: Analysis of reprogramming in yolk sacs from Prox1 double transgenic embryos. (A) Analysis of control and DT embryos at E10.5 (lanes 1 and 2) and yolk sacs at E10.5 or E12.5 (lanes 3 to 6) for prox1 expression by RT-PCR. (B) Yolk sacs from E13.5 control and DT embryos were analyzed using RT-PCR for the expression of lymphatic associated markers such as CyclinE2, Stat6, VEGFR-2, VEGFR-3 and Neuropilin-1 (NRP1). -ve represents no template control. (C) Similarly, yolk sacs from E12.5 control and DT embryos were analyzed by western blot for the expression of VEGFR-2, VEGFR-3, Tie2 and Neuropilin-2 (NRP2).
Mentions: Given the influence of Prox1 on gene regulation in vascular and lymphatic endothelial cells, we further investigated the possibility of a switch in the molecular identity of endothelial cells using markers that are influenced by Prox1. To this end, we started our analysis with the yolk sac, a relatively simple tissue that is rich in blood vasculature (Figure 3a). Analysis of control or DT embryos reveal that the overexpression of Prox1 in vascular endothelial cells could alter gene expression to that of a more lymphatic endothelial cell signature. For example, transcripts for genes such as VEGFR-3 and CyclinE2 increase while VEGFR-2, Neuropilin1 and Stat6 decrease (Figure 3b). Furthermore, protein levels from yolk sacs derived from DT embryos demonstrate that VEGFR-2 and Tie2 decrease while Neuropilin-2 increases with a marginal increase in VEGFR-3. Wholemount analysis of DT yolk sacs support the reprogramming event, indicated with an increase in Podoplanin expression that correlates with Prox1 overexpression (data not shown). Consistent with previous findings, the overexpression of Prox1 in vascular endothelial cells appear to sufficiently alter the gene signature of BECs to that of a more LEC profile in vivo.

Bottom Line: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature.Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sunnybrook Research Institute University of Toronto 2075 Bayview Avenue Toronto, Ontario M4N 3M5, Canada. dan.dumont@sri.utoronto.ca

ABSTRACT

Background: In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo.

Results: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.

Conclusion: We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

Show MeSH
Related in: MedlinePlus