DEV02220 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effect of cyclopamine on development of cultured embryonic kidney explants. Hematoxylin stained 4 mm histological sections were generated from wild-type kidneys cultured from E11.5 in the presence of drug vehicle alone (100% ethanol in culture medium) or cyclopamine for 4 days. Kidneys treated with drug vehicle alone demonstrated formation of glomeruli precursors (A) and, at higher resolution, formation of ureteric bud branches and epithelial metanephric derivatives (B). By contrast, cyclopamine interrupted formation of these elements resulting in tissue disorganization and dilated tubules (C,D). Scale bar: 100 mm.

• Supplemental Figure 2 -

  Fig. S2. PCR products identified by agarose electrophoresis after chromatin immunoprecipitation. Analysis of Shh target genes. In embryonic kidneys isolated at E13.5, GLI1 and/or GLI2 bind 5¢ flanking regions containing GLI consensus binding regions in Pax2, Sall1, cyclin D1 and Mycn. No binding of GLI3 was detectable. Neg, DNA amplified after immunoprecipitation with non-immune serum; Pos, DNA amplified using genomic DNA and specific primers; Input, DNA amplified from cross-linked DNA before immunoprecipitation.

• Supplemental Figure 3 -

  Fig. S3. PCR products identified by agarose electrophoresis after chromatin immunoprecipitation. Analysis of Gli1 and Gli2. In embryonic kidneys isolated at E13.5, GLI2 binds a 5¢ flanking region containing a GLI consensus binding region in Gli1. No binding of GLI3 was detectable. A similar region in Gli2 was bound by GLI1, GLI2 and GLI3. NS, DNA amplified after immunoprecipitation with non-immune serum; Pos, DNA amplified using genomic DNA and specific primers; Input, DNA amplified from cross-linked DNA before immunoprecipitation.

• Supplemental Figure 4 -

  Fig. S4. Agarose gel electrophoresis of products generated by RT-PCR using RNA isolated from cultured embryonic kidneys treated with or without cyclopamine. Cyclopamine decreased expression of mRNAs encoding Gli1, Gli2 and Pax2, but exerted no effect on Gli3 mRNA. V, vehicle; C, cyclopamine