DEV020586 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. WNT ligand expression in the developing GT. (A-F) ³⁵S in situ hybridization on E12.5 coronal sections of GT using probes indicated; signals are red and nuclei are blue. Wnt2 was expressed in the lateral region of proximal GT mesenchyme (arrows, A). Wnt3 was expressed in the surface ectoderm (arrows, B). Wnt5a expression was detected in distal mesenchyme and distal urethral epithelium, whereas Wnt11 expression was detected in proximal mesenchyme and the proximal urethra (D). Wnt9b transcripts were detected in the urethral epithelium and the surrounding mesenchyme (E). Lef1 was expressed exclusively in the distal GT mesenchyme (F). Scale bars: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Colocalization of TOPGAL expression and Fgf8 expression. (A,C,E) Whole-mount X-Gal staining of E10.5 (A,E), and E12.5 (C) embryos carrying the TOPGAL transgene, note the dUE staining (A,C) and the AER staining (E). (B,D,F) Whole-mount Fgf8 in situ hybridization showed dUE (B,D) and AER (F) Fgf8 expression. The arrows indicate TOPGAL-positive cells and Fgf8-expressing cells colocalized in the dUE and the AER.

• Supplemental Figure S3 -

  Fig. S3. Altered gene expression and apoptosis in the endodermal β-catenin mutants. (A-E′′) Whole-mount in situ analysis using probes indicated on E12.5 control (A-E), Shh^Cre/esr;β-Cat^c/c (LOF, A′-E′) and Shh^Cre/esr; β-Cat^loxEx3(GOF, A′′-E′′) GTs. All embryos were exposed to Tm on E10.5. (A-A′′) Shh was expressed in the UE (A), the expression was downregulated in the UE of LOF (A′, arrows), and also downregulated in the distal UE of GOF GT (A′′, arrows). The proximal UE Shh expression in GOF GT was not disturbed (A′′, arrowheads). (B-B′′) Ptc1 was expressed in mesenchymal cells surrounding Shh-expressing UE (B), the expression was downregulated in LOF GT (B′, arrows), and in distal mesenchyme of GOF GT (B′′, arrows). (C-C′′) Phosphorylated SMAD1/5/8 immunostaining revealing downregulation of pSMAD1/5/8 expression in the distal mesenchyme of LOF GT (C′) and upregulation of pSMAD expression in the UE of GOF GTs (C′′). (D-D′′) TUNEL assay revealed no difference in the number of apoptotic cells in both LOF and GOF urethra, and reduced distal mesenchymal apoptosis in the LOF GTs (C′). (E-E′′) Whole-mount in situ analysis showed reduced Fgfr2 expression in the urethral epithelium in both LOF (E′) and GOF (E′′) GT. (F) Real-time RT-PCR analysis showed a 30% reduction in Fgfr2 transcripts in LOF GTs (P=0.0041). The expression in GOF GTs appeared to be lower than that in wild type. However, no significant difference was detected (P=0.058). Thus, these data were still not conclusive. For real-time analysis, three pools of GT RNA each isolated from eight GTs were used. Scale bars: 50 µm in D-D′′.

• Supplemental Figure S4 -

  Fig. S4. Histological and molecular analysis of defective ectodermal and endodermal urethra in Msx2-Cre; β-Catc/c GT. (A,B) Hematoxylin and Eosin-stained sagittal sections (illustrated in F) of E12.5 control (A) and Msx2-Cre; β-Catc/c (B) GT. Note the disorganized distal urethral plate in the mutant (B, arrows). X-Gal-stained sagittal section of E12.5 Msx2-Cre;R26R GT showed universal Cre activity in the ectodermal-derived surface epithelium (inset in B). (C,D) Immunohistochemistry against β-catenin demonstrated complete removal of β-catenin in the ectoderm (D, arrows) and exposure of β-catenin-positive UE cells on the distal surface of Msx2-Cre; β -Catc/c GT (D, arrowheads) (E,F) Whole-mount X-Gal staining showed that TOPGAL expression in Msx2-Cre; β-Catc/c GT was shifted more distally and was expanded on the distal top (F, arrow). (G,H) Consistently, Fgf8 expression assayed by whole-mount in situ hybridization was also shifted (H). However, the expression levels and the number of cells expressing Fgf8 were not changed. Scale bars: 100 µm.

• Supplemental Figure S5 -

  Fig. S5. Phenotype of Dermo1-Cre; β-Catc/c GTs. (A-F) SEM analysis on E13.5, E14.5 and E16.5 embryos revealed hypoplastic and dysmorphic GTs in Dermo1-Cre; β-Catc/c embryos (B,D,F compared with A,C,E). (G-L) Double immunofluorescence against β-catenin and the mitotic marker PHH3 demonstrated partial deletion of β-catenin (H, arrows indicate β-catenin-positive region), reduced proliferation (J compared with I) and the correlation of β-catenin deletion with reduced proliferation (L, note the dense red-stained PHH3-positive cells were localized with the green-stained β-catenin-positive cells) in the mesenchyme of Dermo1-Cre; β-Catc/c GTs. (M,N) In situ hybridization showed downregulation of cyclin D1 in the Dermo1-Cre; β-Catc/c GT mesenchyme (N). (O) Bar chart showing a more than twofold reduction in the percentage of PHH3-positive cells in selected regions (shown in I and J). Scale bars: 100 µm in G-N.

• Supplemental Figure S6 -

  Fig. S6. Phenotype of E18 Msx2-Cre; β-Cat^c/c GT in males and females. (A-D) H&E staining of transverse sections of wild-type and Msx2-Cre; β-Cat^c/c GTs, the genotype and sex of the embryos are indicated in the figure. (E-H) Immunofluorescence using a monoclonal antibody against β-catenin. Note that the β-catenin-positive urethral epithelial cells (white arrowheads) are exposed in both male (G) and female (H) mutant GTs.