Lymphatic deletion of Calcrl leads to developmentally arrested lymphatics, hypoplastic jugular lymph sacs, and dilated dermal lymphatic vessels. (A) Measurement of jugular lymph sac area in E14.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos. Quantitative data are represented as mean and SEM for area. n = 6–7 animals per group. (B) Representative images of H&E-stained sections of E14.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos showing jugular lymph sac (JLS), jugular vein (JV), and severe edema (*). Bars, 100 µm. (C) Number of Ki67-positive cells per jugular lymph sac in Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos. Quantitative data are represented as mean and SEM for number of Ki67-positive cells. n = 6 animals per group. (D) Representative images of LYVE1 (green), PROX1 (red), and Ki67 (blue) stained sections of E14.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos showing proliferating cells in jugular lymph sac (JLS). Boxed regions are shown as magnified insets. Arrowheads highlight Ki67-positive lymphatic endothelial cells. Bars, 100 µm. (E) Dermal lymphatic vessel diameter of Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos. n = 3–5 animals per group. (F) Podoplanin (PDPN, red) stained dermal lymphatic vessels from E13.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos. White line highlights width of the lymphatic vessel. Bars, 25 µm. (G) Quantification of phosphohistone H3 (pHH3)–positive cells in E13.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 dermal lymphatic vessels. Data are represented as mean and SEM for number of pHH3-positive cells per branch. n = 3–5 animals per group. (H) Representative images of PDPN (red) and pHH3 (green) stained dermal lymphatics of E13.5 Calcrl^fl/fl and Calcrl^fl/fl/Prox1-CreER^T2 embryos. Arrowheads indicate pHH3-positive cells. Bars, 1 µm. Quantitative data are represented as mean and SEM. In all panels, significance was determined by Student t test (tail = 2, type = 2) with *, P < 0.05; **, P < 0.005; ***, P < 0.0001.