Fig. 6. Alk2 is essential for MIS signaling, transition of Misr2 expression and Müllerian duct regression in the rat. E14.5 male urogenital ridges were treated with control-siRNA (A,C,E) or Alk2-siRNA (B,D,F) for 10 hours. (A,B) Cultured for additional 10 hours followed by whole-mount immunofluorescence analysis of activated R-SMAD1, 5, 8 (P-SMAD). (C,D) Cultured for an additional 20 hours followed by in situ hybridization to detect Misr2. (E-H) Cultured for an additional 48 hours followed by in situ hybridization to detect Wnt7a expression. White arrows indicate the cranial regions with high (A) or low (B) P-SMAD expression for comparison. The presence (C) or absence (D) of Misr2 expression can be noted in the regions between the Müllerian and Wolffian ducts (arrowheads). Black arrows indicate the persistence of Wnt7a expression in the remaining Müllerian duct epithelium (F,H). The position of transverse sections (G,H) is marked by broken lines on E and F, respectively. Cranial is oriented towards the top and Müllerian duct to the right of individual images. M, Müllerian duct; T, testis; W, Wolffian duct. Scale bar: 500 µm for all the whole-mount samples; 50 µm for all the sections.