Epo expression analysis in wild-type and Wt1-deficient mouse embryos. (A) Double-immunofluorescence labeling for Wt1 (red) and EPO (green) in sections of the murine embryonic liver (E10.5). Wt1 is expressed predominantly in hepatocytes at the periphery of the liver because the typical lobular architecture at this early developmental stage is not yet established (Ai; higher magnification, Avii; negative control, Aii). Nuclear localization of Wt1 is confirmed by counterstaining with DAPI (Aviii,Aix). Hematoxylin-eosin staining reveals nests of hematopoietic progenitor cells in the developing liver (Av, arrow). Labeling of Epo is shown in Aiii, Avi, and Avii at higher magnification (Aiv, negative control). Note the colocalization of Wt1 and EPO in hepatocytes at the periphery of the liver (Avi, Avii, Aix). (B) Representative RT-PCR results for Epo mRNA expression in the liver of a mouse embryo at E12.0 with homozygous Wt1 deletion (Wt1^-/-) in comparison with a wild-type littermate (Wt1^+/+). Note the reduced Epo mRNA level in the liver of the Wt1-deficient embryo. Gapdh signal revealed a similar efficiency of RNA preparation and cDNA synthesis in both livers. For better visualization, the gel photograph is presented as a negative of the original. M = 100 bp DNA ladder as a marker for the size of PCR products. (C) Real-time RT-PCR analysis of Epo expression in the livers of 9 murine embryos (E12.0) lacking Wt1 (Wt1^–/– ▪) compared with 9 wild-type littermates (Wt1^+/+ □). EPO expression, which was defined as 100% in wild-type embryos, was normalized to β-actin transcripts. For comparison, albumin and α-fetoprotein (Afp) mRNA levels were not significantly different (P > .05) in the liver specimens of wild-type and Wt1-deficient embryos (n = 5 each). Values are mean ± SEM. *Statistical significance (P < .01; Student t test).