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Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene

Patrick D. Brophy^1,*, Lance Ostrom^2,*, Katherine M. Lang² and Gregory R. Dressler^2,

¹ Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA
² Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
^* These authors contributed equally to this work

View larger version (69K): [in a new window]   Fig. 1. Expression of Pax2 in E11.5 metanephric mesenchyme. Whole-mount antibody staining with antibodies against Pax2 (red) and pan-cytokeratin (green) in wild-type (+/+) and Ret-/- mutants as indicated. Pax2 protein is localized to the nephric duct (ND), ureteric bud (UB) and metanephric mesenchyme (MM) in wild-type E11.5 kidneys. Anti-pan-cytokeratin stains the epithelium of the nephric duct and ureteric bud only. Note the expression of Pax2 in the mesenchyme of Ret mutants despite the absence of any ureteric bud. Scale bars: 80 µm.

View larger version (62K): [in a new window]   Fig. 2. Expression of GDNF in Pax2 mutants. (A) Whole-mount in situ hybridization of GDNF probe to E10.5 and E11.5 nephric duct (nd) and metanephric mesenchyme (m). In wild-type embryos, GDNF localizes to the posterior mesenchyme of the E10.5 urogenital region. Note the lack of GDNF staining in Pax2 mutants at both E10.5 and E11.5. (B) RT-PCR from RNAs isolated from E11.5 metanephric mesenchyme; genotypes are as indicated. Serial dilutions of total RNA was used for RT-PCR with GDNF specific primer pairs. Control RT-PCR reactions used primers for the Gapdh gene (GP3DH). Note lack of GDNF-specific PCR product in Pax2-/- mutants, but not in Ret-/- mutants.

View larger version (109K): [in a new window]   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).

View larger version (99K): [in a new window]   Fig. 4. Pax2 mutant mesenchyme is unresponsive to induction. Metanephric mesenchymes (arrows) from Pax2 and Ret homozygous null embryos were isolated at E11 and co-cultured with E11 dorsal spinal cord (sc). (A) Phase contrast micrographs taken at 0, 24 and 72 hours post explant. Note the disappearance of Pax2 mutant mesenchyme, whereas the Ret mesenchyme forms epithelial aggregates within 24 hours. (B) Whole-mount immunostaining for E-cadherin and Pax2 as indicated. Tubules derived from the Ret mesenchyme show prominent expression of E-cadherin. What little remains of the Pax2 mutant mesenchyme is negative for E-cadherin. Scale bars: 500 µm.

View larger version (45K): [in a new window]   Fig. 5. Activation of GDNF mRNA in Pax2-expressing cell lines. (A) Northern blot analysis of parental 46m cells and Pax2 clonal derivatives probed for GDNF, Pax2 and GAPDH. Note increased GDNF mRNA in cells expressing Pax2b cell lines and low level of GDNF mRNA in Pax2a cell lines. (B) Infection of 46m cells with Pax2b expressing adenoviral vector. Top panel shows western blot of total cell lysates 24 hours after infection with increasing multiplicity, expressed as PFU/ml. Bottom panel shows a northern blot of uninfected 46m cells and cells infected with 2x109 PFU/ml at 8, 24 and 48 hours post infection. Note expression of GDNF mRNA in total RNA from infected cells.

View larger version (20K): [in a new window]   Fig. 6. Analysis of GDNF-CAT reporter genes in transfected cells. (A) Schematic of the GDNF genomic locus. Boxes represents exonic sequences; the black vertical bars represent prospective Pax2-binding sites. The reporter plasmid p2.4-CAT contains GDNF sequences extending from unique BamHI site in exon1 to HindIII site approximately 2.4 kb upstream, fused to the CAT gene. This corresponds to position -1260 to +1052, according to previous numbering (Tanaka et al., 2000). Plasmid pApa-CAT has a deletion of 250 bp within the 5' UTR that encompasses the Pax2-binding site PBS2. Plasmid pPBS2-CAT has a small 34 bp deletion around the Pax2-binding site (PBS2). (B) CAT activity of reporter plasmids in transfected NIH 3T3 cells. Reporter plasmids were transfected with increasing amounts of Pax2b and Pax2a expression plasmids. Fold activation was calculated relative to the basal level of reporter gene expression. Data are presented as average values for activation and error bars reflect one s.e.m.

View larger version (69K): [in a new window]   Fig. 7. Identification of Pax2-binding sites on the 2.4 kb GDNF promoter region. (A) Electrophoretic mobility shift experiments using isolated fragments or pools of fragments corresponding to the position of the published sequence as indicated. Increasing amounts (0, 1, 5 µl) of diluted Pax2-PD protein were used. The arrows indicate the shifted species in the fragment -237/-38 (PBS1) and +697/+968 (PBS2). PBS2 is also seen in the pooled fragments +500/+1054 (arrow). (B) Electrophoretic mobility shift experiments using increasing amounts of Pax2 protein and oligonucleotides corresponding to the predicted binding sites, based on DNAseI footprinting experiments. (C) Competition experiment using oligonucleotides for PBS1 and PBS2 and increasing amounts of excess (50x, 500x) unlabeled competitor oligonucleotide. (D) DNAseI footprint of PBS2 using increasing amounts of Pax2-PD bound to the fragment corresponding to the region of +697 to +968. Unbroken black bar indicates position of major protected region, with broken line indicating the extended footprint observed at higher concentrations of Pax2-PD.

View larger version (13K): [in a new window]   Fig. 8 Comparison of Pax2-binding sites. The PBS2 site from the GDNF 5' UTR was compared with known Pax2 DNA-binding sites. The previously described sites include a consensus sequence derived by PCR (Epstein et al., 1994); the consensus sequence from chromatin immunoprecipitation studies (Phelps and Dressler, 1996); the Pax2-binding site from the Pax5 enhancer (Pfeffer et al., 2000); and a Pax2 binding site from the WT1 promoter (McConnell et al., 1997). The alignment was made with the ClustalW feature from the MacVector DNA analysis software (Oxford Molecular Group). The PBS2 sequence corresponds to the position +768 to +807 of the published mouse 5' UTR (Tanaka et al., 2000). The region protected by the Pax2 paired domain is underlined. The gray boxes mark the consensus nucleotides for the set of sequences.