DEV022087 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Schematic representation of the Wnt7b^c3 conditional allele before and after Cre-mediated recombination. Exon 3 of the Wnt7b gene was flanked by loxP sites (‘floxed’) to enable Cre-mediated removal of this essential exon sequence. The truncated protein translated from transcripts from the recombined allele (Wnt7b^d3) was non-functional.

• Supplemental Figure S2 -

  Fig. S2. Urine from Wnt7b mutants is hypo-osmotic. The osmolality was measured in a 10-µl urine sample from wild-type and Wnt7b^c3/−; Hoxb7Cre mutants with a vapor pressure osmometer. The urine of Wnt7b^c3/−; Hoxb7Cre mutant pups at P10 (n=6) was significantly less concentrated than that of their wild-type littermates (n=4).

• Supplemental Figure S3 -

  Fig. S3. Normal branching morphogenesis in Wnt7b mutants. Whole-mount in situ hybridization was performed on E14.5 kidneys to visualize ureteric branch tips through the expression of Wnt11. No difference was observed in cortical branching events when Wnt7b mutants were compared with their wild-type littermates.

• Supplemental Figure S4 -

  Fig. S4. The onset of the failure of renal medulla formation does not result from increased apoptosis of the prospective collecting duct epithelium in Wnt7b mutants. Histogram of apoptotic figures within the forming medullary collecting duct epithelium of Wnt7b mutants compared with the deep nascent collecting duct epithelium of wild-type littermates. A similar incidence of apoptosis was observed at E15.5 in wild-type and Wnt7b mutant kidneys. At E16.5, there was a small but statistically significant decrease in the cell death rate in Wnt7b mutants. n=4, for all stages and genotypes. Five hundred to 1600 cells were counted in each sample.

• Supplemental Figure S5 -

  Fig. S5. Canonical Wnt reporter expression in embryonic kidneys. (A) BAT-gal reporter β-galactosidase (β-gal) immunostaining, red expression was detected in collecting duct epithelium (double positive for cytokeratin, purple, and E-cad, green) and non-epithelial interstitial mesenchyme (arrow). (B) Interstitial cells that produced β-galactosidase were also Lef1 positive. (C) A subset of BAT-gal⁺ cells were also p57Kip2⁺ (arrows). The mosaic expression of the β-galactosidase reporter most likely results from position effect influences on the transgene. The renal pelvis is localized at the bottom of these panels. Scale bar: 20 µm.

• Supplemental Figure S6 -

  Fig. S6. Expression of Cre recombinase from the Foxd1GC transgene leads to the ablation of b-catenin expression in the interstitium. Foxd1GC-descendant interstitial mesenchymal cells were β-galactosidase⁺ (the result of Cre-dependent activation of a Rosa26 reporter allele) and E-cadherin⁻. No β-catenin was detected in these cells following Foxd1GC-driven conditional removal of β-catenin activity. E-cadherin was present in the collecting duct epithelium and the nephron tubule epithelium, except for part of the loop of Henle, the epithelial nature of which is discernible by β-catenin expression patterns (white arrows). Scale bar: 40 µm.

• Supplemental Figure S7 -

  Fig. S7. Analysis of the expression of renal medullary regulators and interstitial mesenchymal Wnt genes on the modulation of Wnt7b signaling and canonical Wnt responsiveness. (A-D) In situ hybridization to Wnt7b mutant kidneys at E15.5. Pod1 was expressed normally in the renal interstitium (A,B), whereas Wnt5a interstitial expression, although present, was slightly reduced (C,D) in Wnt7b mutants. (E,F) Immunostaining of integrin α3 (Itga3) in collecting ducts at E15.5 was similar in Wnt7b mutants to in wild-type kidneys. (G-N) Expression of Wnt11 and Wnt4 was greatly reduced in the renal interstitium of both Wnt7b mutants (H,L) and β-catenin interstitium mutants (J,N). Scale bars: 100 µm in A-D,G-N; 20 µm in E,F.

• Supplemental Figure S8 -

  Fig. S8. Ureter development is normal in Wnt7b mutants. Analysis of kidney ureter function and organization in control (A,B,D,F,H,J,L) and Wnt7b mutants (C,E,G,I,K,M) at E18.5. (A-C) Dyes injected into the renal pelvic space flowed into the ureter (black arrows) and the bladder (blocked arrow) of Wnt7b mutants. (D,E) Dyes injected into the bladder (blocked arrow) did not flow back to the ureter of Wnt7b mutants (black arrows). (F-I) Smooth muscle α-actin (SMAA) was present along the length of the ureter of Wnt7b mutants similar to the distribution in wild-type ureter. (J-M) Ptch1 mRNA was appropriately expressed in ureter mesenchyme, indicating active Shh signaling in smooth muscle progenitors of Wnt7b mutants. Scale bar: 50 µm in F-I; 100 µm in J-M.