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First published online 10 May 2006
doi: 10.1242/dev.02383

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Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression

Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

View larger version (63K): [in a new window]   Fig. 1. MIS signaling and dynamic expression of Misr2, Alk2 and Smad8 in male rat urogenital ridges. The mRNAs of Misr2 (A-D), Alk2 (E-H), and SMAD8 (I-L) were detected by in situ hybridization (whole-mount and cryosection) in the urogenital ridges of E14.25 (A), E14.5 (B,E,I), E14.75 (C,F,J) and E15 (D,G,K). (H,L) Cryosections at the at the level of the broken lines in G and K, respectively. (M-O) Expression of phosphorylated SMADs1, 5, 8 (P-SMAD) was detected by whole-mount immunofluorescence analysis in E14.5 (M), E14.75 (N) and E15 (O) urogenital ridges of male rats. Arrows indicate the first detected substantial expressions. Arrowheads indicate expressions in the coelomic epithelium (CE). 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 (A-C,E-G,I-K,M-O); 50 µm for all the sections (D,H,L).

View larger version (49K): [in a new window]   Fig. 2. Expression patterns of the MIS type I receptors (Alk2 and Alk3), Smad5 and P-SMAD in E15.5 rat urogenital ridges. (A,B) Whole-mount in situ hybridization shows decreased Alk2 expression in the cranial coelomic epithelium (A, arrow) and increased Alk3 expression in the mesenchyme (B, black arrowhead) of male urogenital ridges. White arrowhead indicates the coelomic epithelium. (C-F) Sexually dimorphic expression of P-SMAD detected by immunohistochemistry. (E,F) Higher magnifications of the boxed area around the Müllerian ducts in C,D. Arrowheads in D,F indicate P-Smad in mesenchyme surrounding Müllerian duct. (G,H) Smad5 expression detected in the coelomic epithelium (indicated by arrows) adjacent to the Müllerian duct of female (G) but not male (H) urogenital ridges. Cranial is oriented towards the top and Müllerian duct to the right of individual images (A,B). M, Müllerian duct; Ov, ovary; T, testis; W, Wolffian duct. Scale bar: 500 µm in A-D; 100 µm in E,F; 200 µm in G,H.

View larger version (81K): [in a new window]   Fig. 3. MIS activates R-SMADs 1, 5 and 8 in urogenital ridges in organ culture. (A-E) Female urogential ridges were harvested at E14.5, directly fixed (A) or cultured for 2 hours (B), 6 hours (C), 30 hours (D-F), in the presence (B-D) or absence (E) of MIS. (F) Male urogenital ridges cultured at E14.5 for 30 hours. P-SMAD expression was detected by whole-mount immunofluorescent analysis. Arrows and arrowheads mark the expression of P-SMAD in the areas lateral and medial to the Müllerian duct, respectively. Cranial is oriented towards the top and Müllerian duct to the right of individual images. Ov, ovary; T, testis. Scale bar: 500 µm.

View larger version (47K): [in a new window]   Fig. 4. MIS causes migration of Misr2-expressing coelomic epithelial cells to the Müllerian duct mesenchyme. Hematoxylin and Eosin (HE) staining (A), immunofluorescent analysis of vimentin expression (B) of cranial transverse sections from E14.5 female urogenital ridges. (C-N) Dynamic change of Misr2 expression, CM-DiI localization and laminin-stained basement membrane in cultured female urogenital ridges with or without MIS treatment. Female urogenital ridges were harvested at E14.5, unlabeled or labeled with CM-DiI (D,F,I,L,N), directly fixed (C,D), cultured with no treatment for 20 hours (E-G) or 40 hours (M,N), or treated with MIS for 20 hours (H-J) or 40 hours (K,L). Arrows indicate the coelomic epithelium (CE). White arrowheads (A,B,J) indicate the basement membrane that separates the coelomic epithelium and subjacent mesenchyme. Arrowheads in H-L indicate the extension of Misr2 expression (H,K), CM-DiI detection in the mesenchyme (I,L) and disruption of the basement membrane (J) under the influence of MIS. M, Müllerian duct; W, Wolffian duct. Scale bar: 50 µm for all the sections.

View larger version (97K): [in a new window]   Fig. 5. MIS upregulates SMAD8, downregulates SMAD5 and induces Alk2 and Alk3 expression in urogenital ridges. Male or female urogenital ridges harvested at E14.5 were untreated or treated with MIS in organ culture for 20 hours (A-L), 12 hours (M-P) or 30 hours (Q-T). Transcripts of SMAD8 (A-F), SMAD5 (G-L), ALK2 (M-P) and ALK3 (Q-T) were detected by whole-mount in situ hybridization (A-C,G-I,M,N,Q,R), and their expressions are also shown after cryosectioning (D-F,J-L,O,P,S,T) at the broken lines. Arrows indicate the presence of expression. Cranial is oriented towards the top and Müllerian duct towards the right of individual whole-mount images. M, Müllerian duct; Ov, ovary; T, testis; W, Wolffian duct. Scale bar: 500 µm for all the whole-mount samples and 50 µm for all the cross-sections.

View larger version (54K): [in a new window]   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.

View larger version (67K): [in a new window]   Fig. 7. Smad5 RNAi enhances and Smad8 RNAi inhibits Müllerian duct regression. E14.5 male rat urogenital ridges were treated with control-siRNA (A,C,E), Smad5-siRNA (B,D) or Smad8-siRNA (F) for 12 hours, and subsequently cultured for indicated periods. Whole-mount in situ hybridization was performed to detect Wnt7a expression. Arrowheads or arrows indicate the disappearing and remaining Wnt7a expression, respectively. Cranial is oriented towards the top and Müllerian duct towards the right of individual images. Scale bar: 500 µm.

View larger version (26K): [in a new window]   Fig. 8. A schematic model of MIS actions at the early stage of Müllerian duct regression. Müllerian duct (M) formation and initial MISRII expression (dark blue) in the coelomic epithelium (gray) are similar in male and female urogenital ridges at E13 and early E14. After E14.5, MIS signaling (yellow) becomes functional in the male, driving the MISRII-expressing cells into the area adjacent to the Müllerian duct and eventually around the Müllerian duct at E15.5. This is an epithelial-to-mesenchymal transition. Meanwhile, MIS also upregulates ALK2 and SMAD8 and downregulates SMAD5. These combined activities have roles in Müllerian duct regression, as noted by the smaller Müllerian duct after E15.5, which disappears eventually. At this time, ALK3 and SMAD8, which are highly expressed in the Müllerian duct mesenchyme may mediate MIS signaling and Müllerian duct regression. Expression of MISRII remains in the coelomic epithelium of female urogenital ridges during this period. M, Müllerian duct; W, Wolffian duct.