Postvascular thymus colonization in CCR7/CCR9-deficient mice. (A) Numbers of thymocytes from indicated mice at E16.5, postnatal day 1 (P1), and P14 are plotted. (B) Two-color flow cytometry analysis of indicated thymocytes for CD4 and CD8. Numbers indicate the frequency of cells in each quadrant. (C) E14.5 fetal thymus lobes were organ-cultured, and thymocyte numbers were measured at day 6 of culture. (D) BrdU was pulsed for 2 hours to pregnant mice carrying E16.5 embryos, and the frequency (means ± SE) of BrdU-incorporated embryonic thymocytes was measured by flow cytometry. (E) Frozen sections of indicated embryos were 3-color stained for endothelial CD31 (green, Alexa 568), mesenchymal ER-TR7 (red, Alexa 633), and keratin (blue, FITC). Higher magnification of the vasculatures is shown in the insets. (F) Scanning electron microscopy images of the vascular architecture in normal mouse embryos at the indicated gestational age. Embryos were intravenously microinjected with resin, and the tissues were digested, as described in “Materials and methods.” (G, H) Equal numbers of lineage-negative bone marrow cells from indicated mice were labeled with CFSE and intravenously administered into adult wild-type mice. On day 2 after the administration, the number of CFSE⁺ cells in thymocytes and blood leukocytes was measured by flow cytometry (panel G), and frozen sections of the thymus were analyzed for migrated CFSE⁺ cells (green), CD31⁺ endothelial cells (blue, Alexa 568), and ER-TR5⁺ medullary thymic epithelial cells (red, Alexa 633) (panel H). ^*P < .05; ^***P < .001; NS, not significant.