DEV02773 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Pax3-cre is expressed in the metanephric mesenchyme and its derivatives. E13.5 (A,B) and E16.5 (C) Pax3-cre^tg/+; Rosa26R^tg/+ kidneys stained with X-Gal (blue) and anti-cytokeratin 8 (brown). Scale bar, 0.1 mm.

• Supplemental Figure 2 -

  Fig. S2. Notch2 mutant kidney develops renal tubules despite a flattened papilla and a collapsed pelvis. (A,D) Wild type. (B,C,E) Notch2 mutant. Arrow and star in A indicate the papilla and the pelvis, respectively. A and B are stained with H&E; C, D, E are stained with anti-cytokeratin 8 (brown, and turquoise arrowhead), and with hematoxylin. Green arrowhead: cytokeratin-8-negative renal tubules. Scale Bar, 0.1mm.

• Supplemental Figure 3 -

  Fig. S3. Pax2-cre^tg is expressed in the urogenital organ. Pax2-cre^tg/+; Rosa26R^tg/+ embryos are stained with X-Gal. (A) Whole-mount staining of the kidney in which the ureter (U), the Wolffian duct (W) and the Mullerian duct (M) are visible. (B) Histology of the X-Gal-stained kidney. Notice the blue mesenchymal cells in the nephrogenic zone, the blue renal epithelia and the blue ducts. Scale bar, 0.1mm. (C,D). No accumulation of NICD was detected in renal epithelia of Pax2-cre^tg/+; N1^f/Δ1 embryos; S-shaped bodies are detected in Pax2-cre^tg/+; N1^f/+ embryos (yellow arrowheads). (E,F,H,K,L) Staining for the full-length Notch1 protein with a rabbit antibody raised against the intracellular domain. (E,F) Wild-type metanephroi in organ culture contain Notch1 in duct and renal epithelia. Basal is indicated in E with a dashed pink line; the arrowhead indicates the apical side. Note the baso-lateral and apical distribution of Notch1 in the duct. Apical staining is intensified in renal epithelia; low signal is detected in the nucleus of S-shaped bodies (E). (G-L) Notch1-deficient metanephroi. Pax2-positive ducts (arrowhead) and Pax2, N-CAM-positive renal epithelia are present but no Notch1 staining is detected. (I-L) adjacent sections were labeled with several antibodies (as indicated in the figure). Fiduciary points are marked with asterisk. A jagged1-positive, N-CAM-positive, Pax2-positive, Notch1-negative RV is marked with an arrowhead. Non-specific background is marked with a diamond in K,L.

• Supplemental Figure 4 -

  Fig. S4. Dll-1^LacZ is expressed in the renal vesicle, the S-shaped body and the proximal tubule segments. (A-E) E17 kidneys. (A) Whole-mount X-Gal staining. In B and C the section is also stained for LTL (brown) and with hematoxylin. Note that inside a renal corpuscle the X-Gal-stained cells are the red blood cells in the glomerulus capillary, not the podocytes. The proximal tubule segment that connects the Bowmen’s capsule (S1) is not blue. (D) E-cadherin (brown) partially overlaps with X-Gal staining (blue) in Dll-1^LacZ S-shaped body (arrowhead). (E) The pattern of N1ICD expression is similar to Dll-1^LacZ. Arrow in both D and E indicates podocyte precursors. S1, proximal tubule segment 1; S2, proximal tubule segment 2; S3, proximal tubule segment 3. (F-H) Kidneys from Dll-1^hyp/LacZ embryo (E18) stained for LTL (brown) and with Hematoxylin. Note that few LTL-positive proximal tubules formed but many glomeruli are present, which can be clearly seen when H&E-stained sections are shown side-by-side (G,H). Red arrow, glomerulus; green arrow, proximal tubule; turquoise arrowhead, collecting duct; PT, proximal tubule. Scale bars: A,B,C, 0.1 mm; D,E, 0.05 mm.