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Fig. S1. Metanephric mesenchyme is absent in the COUP-TFII deletion mutant. (A-L) Tam was administered at E7.5 to delete COUP-TFII. Serial transverse sections of metanephric mesenchyme (mm) regions (caudal to rostral) of the embryos were stained with Hematoxylin and Eosin at E10.5. The condensed mm is seen as early as the Wolffian duct (wd) just proximal to its entry into the posterolateral surface of cloaca in the COUP-TFIIf/f control embryo (A). A series of sections including A, C, E, G, I and K showed the appearance of condensed metanephric mesenchyme (mm) in the COUP-TFIIf/f control embryo. However, the metanephric mesenchyme (mm) is completely absent in the comparative sections including B, D, F, H, J and L in the COUP-TFIId/d mutant embryo. Scale bars: 50 µm.
Fig. S2. COUP-TFII is expressed in the mesonephros but is not essential for mesonephros formation. (A) COUP-TFII staining in the transverse section of the E10 (Tam administration at E7.5) COUP-TFIIf/f control embryo showing that COUP-TFII is expressed in the mesonephros (me) and not in the Wolffian duct (wd). (B) Pax2 staining is detected in both mesonephros (me) and Wolffian duct (wd), but only colocalizes with COUP-TFII in the mesonephros (me). (C,D) Pax2 staining results indicated that both mesonephros and Wolffian duct are present in the E9.5 COUP-TFII+/+ control (C) and the COUPTFII−/− mutant (D). Insets show that the expression of COUP-TFII in the mesenchyme cells surrounded the Wolffian duct of the control (C) and the expression of COUP-TFII is ablated in the COUP-TFII−/− mutant (D). Scale bars: 50 µm.
Fig. S3. COUP-TFII colocalizes with Wt1 in the metanephric mesenchyme and urogenital ridge. (A-C) Double staining with COUP-TFII and Wt1 antibodies in the transverse section of the E10.5 (Tam administration at E7.5) COUP-TFIIf/f control embryo. Wt1 is expressed in the metanephric mesenchyme (mm) and urogenital ridge (ur) (B) and COUP-TFII expression is also detected at the same metamephric mesenchyme (mm) and urogenital ridge (C). These results indicate that COUP-TFII colocalizes with Wt1 in the metanephric mesenchyme and in the urogenital ridge, but not in the Wolffian duct (wd). Scale bars: 25 µm.
Fig. S4. COUP-TFII alone can regulate Eya1 and Wt1 gene expression in the metanephric mesenchyme in vitro. Quantitative RT-PCR analysis of COUP-TFII (A), Eya1 (B) and Wt1 (C) in rat metanephric mesenchyme-derived cells (RIMM-18), subsequent to knockdown of COUP-TFII using COUP-TFII-specific siRNAs. All mRNA measurements were normalized to 18S rRNA. Error bars indicate s.d. **P<0.005.
Fig. S5. COUP-TFII and Osr1 signaling pathways work in parallel to promote nephrogenic mesenchyme development. (A,B) In E10.5 embryos (Tam administration at E7.5) Osr1 RNA expression was detected broadly in the mesenchyme region surrounding the Wolffian duct (wd), including metanephric mesenchyme (mm), in both COUPTFIIf/f control (A) and in the CRE-ERT2;COUPTFIId/d mutant embryo (B). (C-F) COUP-TFII expression in the nephrogenic mesenchyme was detected in both E10.5 Osr1wild type (C,E) and Osr1 knockout embryos (D,F). The slides are counterstained with DAPI for nuclei (E,F, blue). Scale bars: 50 µm. (G-L) Quantitative RT-PCR analysis of Osr1 RNA level (I) in the rat metanephric mesenchyme-derived cells, RIMM-18, after knock down of both COUP-TFI and COUP-TFII (with COUP-TFI siRNA and two different COUP-TFII siRNAs; G-I) for 48 hours. The mRNA measurements were normalized to 18S rRNA. In the reverse experiment, COUP-TFI (K) and COUP-TFII (L) expression levels were also examined by quantitative RT-PCR analysis in RIMM-18 cells after knock down of Osr1 expression by siRNA to Osr1. mRNA measurements were normalized to 18S rRNA. Error bars indicate s.d. *P<0.05, **P<0.005.
Fig. S6. Construction of Eya1 and Wt1 wild-type and Sp1-binding site mutant reporters. (A) The 1.8 kb human EYA1 promoter region from −731 to 1079, which contains three conserved Sp1-binding sites, was amplified from human BAC clone RP11-160C3 with paired primers (Eya1-pro-Kpn-5′ and Eya1-pro-Xho-3′; supplementary material Table S4). This fragment was digested with restriction enzymes KpnI and XhoI then cloned into pGL2-basic reporter plasmid (A). The pGL2-Eya1-M1 changed cytidine to adenosine within the first Sp1-binding site located between −298 bp and −286 bp. The pGL2-Eya1-M2 changed cytidine to thymidine within the second Sp1-binding site located between −126 bp and −114 bp. The pGL2-Eya1-M3 changed cytidine to thymidine or adenosine within the third Sp1-binding site located between −40 bp and −23 bp. All the mutated bases are shown in light gray and are underlined. (B) The 1.1 kb human WT1 promoter region from −736 to 377, which contains two conserved Sp1-binding sites was amplified from human BAC clone CTD-2083A15 with paired primers (WT1-pro-KpnI-5′ and WT1-pro- BglII-3′; supplementary material Table S4). This fragment was digested with restriction enzymes KpnI and BglII then cloned into the pGL2-basic reporter plasmid. The pGL2-Wt1-M1 changed cytidine to adenosine within the first Sp1-binding site located between −349 bp and −313 bp. The pGL2-Eya1-M2 changed cytidine to adenosine within the second Sp1-binding site located between −300 bp and −288 bp. All the mutated bases are shown in light gray and are underlined.
Fig. S7. Kidney organ culture of COUP-TFII deletion mutant. (A,B) Kidney organ culture from the metanephric mesenchyme of COUP-TFIId/d mutant embryo shows swelling at the end of the Wolffian duct (arrowhead), which indicates the possibility of ureteric bud outgrowth.
