Figure 1.
Cre recombinase-mediated deletion of floxed sequences in dermal lymphatic vessels.
(A,B) Whole-mount preparations of ear skin from mT/mG and Lyve-1wt/Cre;mT/mG mice. GFP is not expressed in the skin of mT/mG mice (A) but is expressed by lymphatic vessels and macrophages in the skin of Lyve-1wt/Cre;mT/mG mice (B). (C-H) Representative images of transverse sections of ear skin from adult Vegfr2flox/flox mice and Lyve-1wt/Cre;Vegfr2flox/flox mice stained with antibodies against Vegfr2 and Lyve-1. Vegfr2 (C) is expressed by Lyve-1 positive (D) lymphatic vessels in Vegfr2flox/flox mice (E, arrow). In contrast, Vegfr2 (F) expression is lost by Lyve-1 positive (G) lymphatic vessels in Lyve-1wt/Cre;Vegfr2flox/flox mice (H, arrow).
Figure 2.
Lyve-1wt/Cre;Vegfr2flox/flox mice exhibit a deficiency of dermal lymphatic vessels.
(A,B) Whole-mount immunofluorescence staining of ear skin from Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox mice for Lyve-1. (C) Quantitative analysis showing that there are significantly fewer lymphatic branch points in the ear skin of Lyve-1wt/Cre;Vegfr2flox/flox mice (6.042±0.198, n = 6) than Vegr2flox/flox mice (11.29±0.940, n = 6). (D) Lymphatic vessel diameter is not significantly different between Lyve-1wt/Cre;Vegfr2flox/flox (52.65 µm±1.303, n = 6) and Vegfr2flox/flox mice (49.05 µm±1.675, n = 6). *** indicates P || 0.001.
Figure 3.
Lymphatic maturation and blood vessel patterning are normal in Lyve-1wt/Cre;Vegfr2flox/flox mice.
Whole-mount immunofluorescence staining of ear skin from adult Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox mice for Lyve-1 (A,B), smooth muscle actin (A,B), and CD31 (C-F). (A,B) Lyve-1 positive lymphatic vessels are not covered by SMA positive cells in Vegfr2flox/flox or Lyve-1wt/Cre;Vegfr2flox/flox mice. (C,D) CD31 expressing lymphatic valves are present in the collecting vessels of Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox mice. (E,F) The patterning of CD31 labeled blood vessels is similar for Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox mice.
Figure 4.
Lymphatic drainage is normal in Lyve-1wt/Cre;Vegfr2flox/flox mice.
(A,B) Intradermally administered EBD is transported from the hind paws to the iliac lymph nodes in all Vegfr2flox/flox (n = 6) and Lyve-1wt/Cre;Vegfr2flox/flox mice (n = 6).
Figure 5.
Lyve1wt/Cre;Vegfr2flox/flox embryos exhibit lymphatic defects.
(A,B) Immunofluorescence staining for Lyve-1 showing jugular lymph sacs in E14.5 Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox embryos. (C) Graph showing that lymph sac area is not different between Vegfr2flox/flox (380,110±76,279 pixels2; n = 8) and Lyve-1wt/Cre;Vegfr2flox/flox embryos (287,350±59,931 pixels2; n = 5). (D,E) Images of back skin from E14.5 Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/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;Vegfr2flox/flox embryos (11.63±0.5239; n = 3) than in Vegfr2flox/flox embryos (19.37±0.5783; n = 3). (G) At E14.5, lymphatic vessel diameter is not significantly different between Lyve-1wt/Cre;Vegfr2flox/flox (26.00±5.859 pixels; n = 3) and Vegfr2flox/flox embryos (23.75±1.652 pixels; n = 4). (H) Graph showing that there are fewer proliferating lymphatic endothelial cells in Lyve-1wt/Cre;Vegfr2flox/flox embryos than in Vegfr2flox/flox embryos at E14.5 and E16.5. At E14.5, 19.45% of LECs in Vegfr2flox/flox embryos (n = 4) were phospho-Histone H3-positive whereas 9.73% of LECs in Lyve-1wt/Cre;Vegfr2flox/flox embryos (n = 4) were phospho-histone H3-positive. At E16.5, 26.94% of LECs in Vegfr2flox/flox embryos (n = 5) were phospho-Histone H3-positive whereas 13.69% of LECs in Lyve-1wt/Cre;Vegfr2flox/flox embryos (n = 7) were phospho-histone H3-positive. (I) Graph showing the percent of viable Lyve-1wt/Cre;Vegfr2flox/flox mice at different developmental stages. (J-M) Images of non-edematous E14.5 (J,K), E16.5 (L) and E18.5 (M) Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox embryos. ** indicates P || 0.01; *** indicates P || 0.001.
Figure 6.
The density of blood vessels is reduced in the yolk sac, liver and lung in Lyve-1wt/Cre;Vegfr2flox/flox embryos.
(A,B) Whole-mount immunofluorescence staining of yolk sacs from E12.5 Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox embryos for CD31. (C) There are significantly fewer blood vessel branch points in yolk sacs from Lyve-1wt/Cre;Vegfr2flox/flox embryos (41.06±3.662, n = 4 embryos) than Vegfr2flox/flox embryos (106.3±3.157, n = 3 embryos). (D-F) Immunohistochemical staining of E14.5 livers for endomucin showing significantly fewer blood vessels in Lyve-1wt/Cre;Vegfr2flox/flox embryos (27.00±4.263, n = 4 embryos) than in Vegfr2flox/flox embryos (45.88±2.850, n = 4 embryos). (G-I) Immunofluorescence staining of E16.5 lungs for endomucin (red) and DAPI (blue) showing that the density of blood vessels is lower in Lyve-1wt/Cre;Vegfr2flox/flox (18.67±0.962; n = 5) embryos than in Vegfr2flox/flox embryos (28.89±1.145; n = 5). * indicates P || 0.05; *** indicates P || 0.001; **** indicates P || 0.0001.
Table 1.
Observed number of mice following crosses between Vegfr2flox/flox and Lyve-1wt/Cre;Vegfr2flox/flox mice.
Figure 7.
Deleting Vegfr2 in the myeloid lineage does not affect the development of the lymphatic vascular system.
(A,B) Whole-mount immunofluorescence staining of ear skin from Vegfr2flox/flox and LysMwt/Cre;Vegfr2flox/flox mice for Lyve-1. (C) Quantitative analysis showing that the number of lymphatic branch points in the ear skin of Vegfr2flox/flox (8.550±0.278, n = 5 mice) and LysMwt/Cre;Vegfr2flox/flox mice (9.150±0.5895, n = 5 mice) are not significantly different from one another. (D) Furthermore, lymphatic vessel diameter is not significantly different between Vegfr2flox/flox (52.98 µm±1.328, n = 5 mice) and LysMwt/Cre;Vegfr2flox/flox mice (50.05 µm±2.031, n = 4 mice).