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. 2007 Dec 20;118(1):40–50. doi: 10.1172/JCI33302

Figure 7. AM signaling is required for the proliferation and growth of jugular lymphatic vessels during embryonic development.

Figure 7

(A–C) The percentage of proliferating cells relative to total cells was determined in the jugular vein and neighboring jugular lymph sac of wild-type (gray bars) and calcrl–/–, AM–/–, and RAMP2–/– (black bars, A–C, respectively) littermate embryos. Percent proliferative cells was defined as the number of BrdU-positive endothelial cells divided by the total number of endothelial cells in each vessel. Error bars indicate SEM. *P = 0.01 for calcrl–/– (A), P = 0.004 for AM–/– (B), and P = 0.01 for RAMP2–/– (C); Student’s t test. n > 6 animals per genotype. (D and E) Whole mount immunofluorescence of developing vasculature identified by PECAM staining and visualized by 3D OPT of wild-type (D) and RAMP2–/– (E) littermate embryos at E13.5. (F and G) Whole mount immunofluorescence of developing lymphatic vasculature identified by VEGFR3 staining and visualized by 3D OPT of wild-type (F) and RAMP2–/– (G) littermate embryos at E14.5. Compare the presence of well-formed jugular lymph sacs in the wild-type embryo (yellow arrows in F) with the relative lack of jugular lymphatic vessels in the RAMP2–/– littermate (G). Note that the retroperitoneal lymph vessel (red arrows) and dermal lymphatic vessels (green asterisks) of RAMP2–/– mice appear normal compared with those of wild-type embryos. Three embryos from 2 different litters were stained for PECAM and VEGFR3. The online supplemental data contains movies for enhanced, high-resolution, 3D viewing (Supplemental Figures 4 and 5). Scale bar: 2,000 μM.