Fig. 3. GDNF promotes ectopic ureteric bud outgrowth. (A-C) Intermediate mesoderm derivatives, including the nephric duct, mesonephros and metanephric anlagen were dissected out at E10.5 and placed on transwell filters. Heparin acrylamide beads (*) were soaked in GDNF and placed along the midline. After 24 hours in culture, tissues were stained with antibodies against Pax2 (red) and pan-cytokeratin (green). (A) Wild-type culture shows ectopic ureteric buds growing towards the midline (arrowheads) only in the posterior half of the culture. Normal position of the metanephros (m) is indicated. Control beads soaked in BSA show no ectopic ureteric buds (data not shown). (B) Pax2 mutant cultures exhibit nephric ducts but do not show ureteric bud outgrowth in response to GDNF beads. (C) A combination of BMP4 and GDNF on the beads inhibits ureteric bud outgrowth in wild-type nephric ducts. (D-F) Whole mount in situ hybridization for RET expression at E10.5; the Pax2 genotype is indicated. (D) In wild-type E10.5 embryos, RET is expressed along the entire nephric duct (arrowhead) and strongly in the developing ureteric bud (arrow). (E) Pax2 heterozygous E10.5 embryos show RET expression similar to wild-type. (F) Pax2-null E10.5 embryos show RET expression in the more anterior nephric duct (arrowhead) and patchy expression more posteriorly (arrow). (G) E9.5 mutant and wild-type embryos show nearly equivalent RET expression in the nephric ducts (arrows). For micrographs (A-F), anterior is upwards. Scale bars: 500 µm (bar in D applies to D-F).
