VEGF-C maintains α4-integrin expression in the erythroid lineage. (A) Staining of β-Gal (VEGF-C) expressing c-kit⁺ and Ter-119⁺ cells in E9.5 YS or E10.5 FL. Bar represents, 10 μm. (B) Relative Vegfc RNA levels in isolated YS ECs (PECAM-1⁺CD41⁻) and isolated YS HSPCs (CD41⁺c-kit⁺). (C) Flow cytometry showing β-Gal (VEGF-C) expression in the E14.5 FL from Vegfc^+/lacZ mice. Of β-Gal⁺ total FL cells, ∼11% are erythroid cells (Ter-119⁺CD45⁻), ∼11% are Ter-119⁺CD45⁺, and 18.2% are nonerythroid hematopoietic cells (Ter-119⁻CD45⁺). (D-E) Flow cytometric analysis and quantification of mean fluorescent intensity (MFI) of integrin α4 expression in CD41⁺c-kit⁺ HSPCs, CD71^midTer-119⁻, and CD71^hiTer-119⁺ cells in the E10.5 YS. Note that the expression level of α4-integrin in the CD41⁺c-kit⁺ HSPCs, CD71^midTer-119⁻, and CD71^hiTer-119⁺ cells is significantly decreased upon Vegfc deletion (n = 3-4 per group; mean ± SEM; *P < .05; **P < .01). (F) qPCR analyses of Itga4, Itgb1, and Itga5 in WT and Vegfc deleted embryos and YS at E10.5. RNA fold changes are shown. Note that Itga4 is significantly decreased in Vegfc deleted YS (n = 5-7 per group; mean ± SEM; *P < .05; **P < .01).