DEV022442 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. E18.5 kidneys. (A) Haematoxylin and Eosin-stained sections from wild-type and Tshz3^lacZ/lacZ kidney at E18.5. The kidney cortex is grossly similar in the wild type and null mutant. In both genotypes, glomeruli had capillary loops. (B) In situ hybridization analysis of Foxd1 and Raldh2, and immunohistochemistry for Pax2 and podoplanin. (C) Immunohistochemistry analysis for Pax2, SMA, aquaporin and uromodulin. In the medulla (arrow) and in the arteries (double arrowhead), SMA staining was normal in both genotypes, whereas no SMAA was detected in the proximal mutant ureter, in contrast to wild-type ureter (arrowhead).

• Supplemental Figure S2 -

  Fig. S2. Bladder and EM. (A,B) Bladder sections stained with Masson's trichrome and Haematoxylin (M&H), which results in a red colour for muscles, blue for nuclei and a blue/green for collagens (epithelia also appear red). (C-F) E15.5 (C,D) and E18.5 (E,F) smooth muscle alpha-actin (SMAA) staining of the embryonic bladder. (G,H) Transmission electron microscopy of E15.5 proximal ureters reveals normal mesenchymal cell condensation both in wild type (G) and Tshz3 mutant (H). Scale bar in G,H: 2µm.

• Supplemental Figure S3 -

  Fig. S3. Cell turnover in the proximal ureter. (A,B) PCNA immunohistochemistry (brown nuclear signal) of a wild-type (+/+; A) and a Tshz3 null mutant (−/−; B) proximal ureter. Note there is no loss of PCNA-expressing cells in either the mutant urothelium or the surrounding mesenchymal layers. (C,D) β-galactosidase-expressing cells (red) around the ureteric urothelium in a heterozygous (C) and a null mutant (D) embryo. There is no loss of periurothelial cells in the null mutant ureter, which expresses lacZ inserted into the Tshz3 locus.

• Movie 1 -

  Movie 1. Wild-type ureter. Contraction initiated approximately a fifth of the way down the ureter, then the most proximal part contracted and was followed by distal propagation. Proximal ureter is on the right side of the movie. The timelapse imaging of ureters was performed after 5 days of culture on an inverted Axiovert 200M (Zeiss) equipped with a spinning-disc confocal head (Perkin Elmer), using a 5×, 0.16 NA objective (Carl Zeiss). Images were acquired every 0.5 seconds for 2 minutes by using a cooled, 14-bit CCD camera (CoolSnap HQ; Roper Scientific, Trenton, NJ) operated by Metamorph Image Software (Universal Imaging Corporation, West Chester, PA). The QuickTime movie is played at approximately four times actual speed.

• Movie 2 -

  Movie 2. Tshz3^−/− ureter. Contraction initiated approximately a fifth of the way down the ureter and, as in wild type, was followed by distal propagation. Significantly, however, the proximal ureter completely failed to contract. Proximal ureter is on the right side of the movie. The timelapse imaging of ureters was performed after 5 days of culture on an inverted Axiovert 200M (Zeiss) equipped with a spinning-disc confocal head (Perkin Elmer) using a 5×, 0.16 NA objective (Carl Zeiss). Images were acquired every 0.5 seconds for 2 minutes by using a cooled, 14-bit CCD camera (CoolSnap HQ; Roper Scientific, Trenton, NJ) operated by Metamorph Image Software (Universal Imaging Corporation, West Chester, PA). The QuickTime movie is played at approximately four times actual speed.