Fig. 8. Reduced expression of the multifunctional zinc-finger protein Wt1 in spleen mesothelium of Barx1^-/- mice. (A) Among the genes previously implicated in spleen development, only expression of Wt1 is readily recognized in the mesothelial lining (arrowheads) of wild-type E13.5 stomach (St) and spleen (Sp). (B) Higher magnification images of Wt1 mRNA expression indicate enrichment in wild-type spleen capsule (the boxed area in the left panel is shown at even higher magnification in the middle panel, where arrowheads point to the outer surface) and substantially reduced levels in Barx1^-/- spleen (right-hand panel). (C) Independent confirmation of reduced Wt1 transcript levels, assessed by RT-PCR on RNA extracted from isolated E13.5 spleen and surrounding mesothelium. Products of conventional RT-PCR are shown in the gel on the left, and quantitation by real-time PCR using different primers in the bar chart on the right. For quantitative RT-PCR, values were first normalized for Gapdh expression and then to a value of 1.0 for control samples (+/? refers to +/+ or +/-). (D) Confirmation of reduced Wt1 expression by immunohistochemistry. Control (+/+) E13.5 stomach and spleen are shown in the left-hand panels and mutant tissues in the right-hand panels; the boxed areas in the upper panels are shown at higher magnification in the lower panels. Arrowheads point to the mesothelium, where Wt1 levels are reduced in Barx1^-/- embryos. St, stomach; Sp, spleen; Pa, pancreas; Kd, kidney; Lv, liver. (E) Wt1 expression in Barx1^-/- E13.5 kidney is intact.