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. 2013 Sep 2;8(9):e74686. doi: 10.1371/journal.pone.0074686

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© 2013 Dellinger et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A,B) Immunofluorescence staining for Lyve-1 showing jugular lymph sacs in E14.5 Vegfr2^flox/flox and Lyve-1^wt/Cre;Vegfr2^flox/flox embryos. (C) Graph showing that lymph sac area is not different between Vegfr2^flox/flox (380,110±76,279 pixels²; n = 8) and Lyve-1^wt/Cre;Vegfr2^flox/flox embryos (287,350±59,931 pixels²; n = 5). (D,E) Images of back skin from E14.5 Vegfr2^flox/flox and Lyve-1^wt/Cre;Vegfr2^flox/flox embryos stained with antibodies against podoplanin (green) and phospho-histone H3 (red). (F) At E14.5, there are significantly fewer lymphatic branch points in Lyve-^wt/Cre;Vegfr2^flox/flox embryos (11.63±0.5239; n = 3) than in Vegfr2^flox/flox embryos (19.37±0.5783; n = 3). (G) At E14.5, lymphatic vessel diameter is not significantly different between Lyve-1^wt/Cre;Vegfr2^flox/flox (26.00±5.859 pixels; n = 3) and Vegfr2^flox/flox embryos (23.75±1.652 pixels; n = 4). (H) Graph showing that there are fewer proliferating lymphatic endothelial cells in Lyve-1^wt/Cre;Vegfr2^flox/flox embryos than in Vegfr2^flox/flox embryos at E14.5 and E16.5. At E14.5, 19.45% of LECs in Vegfr2^flox/flox embryos (n = 4) were phospho-Histone H3-positive whereas 9.73% of LECs in Lyve-1^wt/Cre;Vegfr2^flox/flox embryos (n = 4) were phospho-histone H3-positive. At E16.5, 26.94% of LECs in Vegfr2^flox/flox embryos (n = 5) were phospho-Histone H3-positive whereas 13.69% of LECs in Lyve-1^wt/Cre;Vegfr2^flox/flox embryos (n = 7) were phospho-histone H3-positive. (I) Graph showing the percent of viable Lyve-1^wt/Cre;Vegfr2^flox/flox mice at different developmental stages. (J-M) Images of non-edematous E14.5 (J,K), E16.5 (L) and E18.5 (M) Vegfr2^flox/flox and Lyve-1^wt/Cre;Vegfr2^flox/flox embryos. ** indicates P || 0.01; *** indicates P || 0.001.