Erythroid-specific Vegf₁₆₄ overexpression affects fetal liver erythropoiesis. (A) Breeding scheme to generate +VEGF164^Eryth-Dox embryos expressing the inducible transcriptional activator rtTA and EGFP constitutively in erythroid cell lineages. Only upon doxycycline administration is Vegf overexpressed via the activation of tet(o)-responsive element by rtTA. Control embryos were littermates lacking the tet(o)-Vegf164 or the EpoR-iCre transgene but receiving doxycycline. (B-C) Relative Vegf mRNA levels measured by qRT-PCR (B) and protein levels measured by ELISA (C) in E14.5 fetal livers after 2 days of doxycycline treatment. (D-F) Doxycycline-induced Vegf164 expression after 2 days of treatment leads to (D) decreased RBCs in the yolk sac, (E) decreased hematocrit, and (F) increased numbers of nucleated RBC progenitors in peripheral blood smears (magnification, 400×) in E14.5 embryos. (G) Definitive erythroid colonies (BFU-E and CFU-E) from E14.5 fetal livers of control and +VEGF164^Eryth-Dox measured by methylcellulose assay after 2 days of doxycycline treatment. Cells (2 × 10⁴) were plated and results are given as percentage to control absolute number of colonies (100%). (H) Relative Gata1, Gata2, Fog1, and Tal1 mRNA levels measured by qRT-PCR in E14.5 fetal livers. Bars in panels B and C and E through H represent mean ± SEM; *P < .05, **P < .01.