Fig. 6. BMP4 signaling is required for the differentiation of the distal UB into the ureter. (A) The Cre-flox system was used to conditionally remove exons 3 and 4 of the Bmp4 gene. The presence of the loxP sites flanking the Bmp4 allele and the occurrence of the recombination event were confirmed through genotyping using specific PCR primers. (B,C) In situ analysis of Bmp4 expression was performed on rudiments isolated from E12.5 embryos and cultured for 4 days. Expression of Bmp4 mRNA is detected in the Bmp4^flox/flox rudiments (B), whereas no Bmp4 is detectable in Bmp4^flox/flox;Cre-Esr1 mutant rudiments (C). (D-K) Metanephric rudiments from E12.5 Bmp4^flox/flox or Bmp4^flox/flox;Cre-Esr1 mutant embryos were cultured for 4 days with 4-OH tamoxifen. Rudiments were analyzed as whole mounts using E-cadherin expression to visualize the UB network (red) and uroplakin (green, D-G) or SMA (green, H-K) to detect ureter differentiation. Uroplakin expression can readily be detected in Bmp4^flox/flox cultures (D,F), but is difficult to detect in the Bmp4^flox/flox;Cre-Esr1 rudiments (E,G). Bmp4^flox/flox rudiments develop an SMA-positive coat (H,J) that condenses around the distal domain of the UB. In the Bmp4^flox/flox;Cre-Esr1 rudiments, only a few unorganized SMA-positive cells are detected (I,K).