DEV039438 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effect of TM on expression of Wnt5a, Dlx5 and Pitx1. (A-F) Expression pattern of Wnt5a, Dlx5 and Pitx1 in control (A-C) and Shh^CreERT2/lox (D-F) GTs treated with TM at E9.75. Note the loss of Dlx5 (B,E) and of Wnt5a (A,D) in the mutants at E13.5, but the preservation of dorsally expressed Pitx1 (C,F).

• Supplemental Figure S2 -

  Fig. S2. Activity of the TopGAL reporter in the GT. (A-D) TopGAL reporter activity in the GT at E12.5 (A), E13.5 (B,D) and E14.5 (C). Note the prominent activity in the distal GT region, including the DUE and part of the urethral plate (arrowheads). (D,D′) Sagittal section of TopGAL reporter mice at E13.5.

• Supplemental Figure S3 -

  Fig. S3. Activity of the BatGAL reporter. (A-D) BatGAL reporter activity (arrowheads) at E11.5 (A,C) and E12.5 (B,D) in Shh mutants (C,D) and controls (A,B).

• Supplemental Figure S4 -

  Fig. S4. Expression of cloacal E-cadherin and β-catenin. (A,C) Cloacal E-cadherin expression in control (A) and Shh^CreERT2/lox (C) E11.5 embryos. (B,D) β-catenin expression in control (B) and Shh^CreERT2/lox (D) E11.5 embryos. TM was administered at E10.5. The dashed line indicates endodermal epithelium, including the DUE.

• Supplemental Figure S5 -

  Fig. S5. The murine Fgf8 locus. Gray boxes indicate exons. The blue box indicates the conserved 3′ region, designated CR3 by Beermann (Beermann et al., 2006). Conserved regions between human and mouse are indicated by the pink rVista peaks. Regions assayed by ChIP are underlined in red. Putative Lef1/Tcf1 binding sequences are shown as black vertical bars.

• Supplemental Figure S6 -

  Fig. S6. Ectopic Fgf4 expression by a loss of Fgf8. (A) lacZ staining in Hoxa3-Cre;R26R reporter mice at E10.5. (B,C) Ablation of Fgf8 mRNA in Hoxa3-Cre;Fgf8^lox/lox mutant (C) relative to control (B) assayed with an Fgf8 probe specific to the floxed exon 5. Arrowhead in B indicates Fgf8 expression in the DUE. (D) Fgf4 expression is not detected in the control GT or DUE. (E) Loss of Fgf8 causes ectopic Fgf4 expression in the distal GT region in Hoxa3-Cre;Fgf8^lox/lox embryos (arrow).

• Supplemental Figure S7 -

  Fig. S7. Expression of Fgf3 in the limb and the GT. (A-F) Fgf3 expression in the GT (A,C,D,F) and limb AER (B,E) of control (A-C) versus Hoxa3-Cre;Fgf8/4^lox/lox (D-F) embryos. In the normal GT, Fgf3 expression is barely detected; however, it is expressed in the distal GT of mutants (white and red arrowheads).

• Supplemental Figure S8 -

  Fig. S8. Effects of constitutively active β-catenin for Fgf10 and Bmp4 expression. (A-F) Expression of mesenchymal genes in control (A,D), Shh KO (B,E) and Shh^CreERT2/−;β-catenin^Ex3/+ (C,F) GTs at E11.5. The levels of mesenchymally expressed Fgf10 (A-C) and Bmp4 (D-F) are decreased in the Shh KO and Shh^CreERT2/−;β-cateninΔ^Ex3/+ embryos. In the Shh^CreERT2/−;β-cateninΔ^Ex3/+ embryos, Bmp4 is ectopically expressed in the endodermal epithelium. Scale bar: 100 µm.