Fig. 4. Differentiation of the TAL, MD and DCT was impaired in the Brn1-deficient kidney. (A) In situ hybridization analyses of the TAL in newborn Brn1^+/+ and Brn1^-/- kidneys. The expression of the Tamm-Horsfall glycoprotein gene (Umod), the prostaglandin E₂ receptor subtype EP₃ gene (Ptger3) and bumetanide-sensitive Na-K- 2Cl co-transporter gene (Nkcc2/Slc12a1) was detected in the TAL of the Brn1^+/+, but not the Brn1^-/- kidney, suggesting that TAL differentiation was impaired in Brn1-deficient animals. Scale bar: 50 µm. (B) Histological and in situ hybridization analyses of the MD and the surrounding TAL cells in Brn1^+/+ and Brn1^-/- kidneys. PAS staining demonstrates that the MD in Brn1^+/+ kidneys (arrows) possesses the defining MD features; cells are crowded and protrude into the tubular lumen. The putative MD in Brn1^-/- mice (arrowheads) does not display these features. Using in situ hybridization analysis, expression of the constitutive type 1 isoform of nitric oxide synthase gene (Nos1) was detected in the MD of the Brn1^+/+, but not the Brn1^-/- kidney. Ptger3 expression was detected in both the MD and the surrounding TAL cells of Brn1^+/+ kidney but was absent from the Brn1^-/- kidney. Scale bar: 20 µm. (C) In situ hybridization analysis of DCT in Brn1^+/+ and Brn1^-/- kidneys. The expression of the thiazide-sensitive Na-Cl co-transporter gene (Ncc/Slc12a3), a maker for DCT, was detected in Brn1^+/+ but not Brn1^-/- kidneys. The expression of the Na/Ca exchanger gene (Ncx1/Slc8a1), a maker of the distal part of the DCT and for the CNT, was altered in Brn1^-/- kidneys in comparison with that of Brn1^+/+ kidneys, suggesting that Ncx1 expression in Brn1^-/- kidneys remained only in the CNT. The expression of the amirolide-sensitive epithelial Na⁺ channel gene (ßEnaC/Scnn1b), a marker for CNT and CD, was indistinguishable in Brn1^+/+ kidney from that in Brn1^-/- kidneys. Scale bars: 20 µm.