Lrf represses expression of Dll4 protein and mRNA in an erythroblast-specific manner. (A) Immunohistochemstry (IHC) of Dll4 in paraffin-embedded BM sections from indicated mice. Magnification: 200× (top row) and 600× (bottom row). Dll4 expression was evident only in mature endothelium of control mice (top, left; black arrowheads), whereas clusters of Dll4-positive cells (red arrowheads) were observed in Lrf knockout and Lrf/Notch1 double-knockout mice. (B) Dll4 expression levels during erythroid differentiation were examined by FACS in control (gray) and Lrf knockout mice (black) 1 month after pIpC injections. Representative FACS histograms of cell surface Dll4 at each differentiation stage are shown. MkP (megakaryocyte progenitor); PreMegE (pre-megakaryocyte–erythrocyte progenitor); CFU-E: colony-forming units-erythroid and FSC (forward scatter: cell size). (C) Dll4 expression levels in myelo/lymphoid lineage cells. PreGM: pre-granulocyte macrophage progenitor and GMP: granulocyte macrophage progenitor. (D) Dll4 mRNA levels were determined by qRT-PCR in FACS-sorted BM erythroid and myeloid cells from control, PHZ-treated control, and Lrf knockout (Zbtb7aFlox/FloxMx1-Cre+, one month after pIpC) mice. Bar graphs show relative expression levels for each fraction. Error bars: standard deviation.
