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First published online 16 November 2005
doi: 10.1242/dev.02095

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Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa

Xiaobo Gao^1,*, Xing Chen^1,, Mary Taglienti¹, Bree Rumballe², Melissa H. Little² and Jordan A. Kreidberg^1,

¹ Department of Medicine, Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
² Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

View larger version (49K): [in a new window]   Fig. 1. Microinjection and electroporation system for kidney organ cultures. (A) A diagram of the microinjection and electroporation system. (a) A three-dimensional depiction, showing a pair of electrodes on the left, and the injection needle on the right. (b) A view from above the organ culture; showing the injection needle placed in the condensed mesenchyme. The dotted line shows the boundary of the whole organ culture. Injections can also be placed outside the condensed mesenchyme, within the looser mesenchyme of the organ culture. (c) Whole-mount double exposure of brightfield image of organ culture and GFP fluorescence 24 hours after injection. (B-E) Gdnf injection/electroporation. (B,D,E) A Gdnf expression vector was injected outside the condensed mesenchyme, adjacent to the Wolffian duct, as shown by the arrow in B. (C) An empty vector was injected as a negative control. (B,C) Cytokeratin stains done 36 hours post-injection to demonstrate the presence of ectopic ureteric buds emerging from the Wolffian duct. Several are present in B, none in C, next to the yellow arrows. (D,E) In situ hybridization for Gdnf, using antisense (D) and sense (E) probes. The endogenous Gdnf mRNA signal is too weak to be detected under these hybridization conditions. CM, condensed mesenchyme; UB, ureteric bud.

View larger version (33K): [in a new window]   Fig. 2. Injection of the Wt1 expression vector. (A) Effect on branching morphogenesis. (a,c,d) Injection of Wt1 cDNA. (b,e) Injection of GFP vector. In situ hybridization with Wt1 antisense probe (a,b), or sense probe (c). The strong area of hybridization is at the single injection site (a), and the endogenous Wt1 signal is observed at several induced nephrons (a,b). (d,e) Cytokeratin staining demonstrating effect of Wt1 on branching. In experiments designed to observe an effect on branching, the vector is injected at two sites, one on either side of the ureteric bud. The branching comparisons shown in each figure are between organ cultures obtained from the same embryo. (f) Overall quantitation of 12 pairs of experiments, represented by d,e. Significant differences in branching were detected after Wt1 injection (P<0.001). (B) Wt1 stimulation of Vegfa expression. Injection of Wt1 (a,c) and Gfp (b) expression vectors. In situ hybridization with Vegfa antisense (a,b) and sense (c) probes. (d) Real-time PCR with Vegfa primers in response to microinjections. As described in the text, a Gfp expression vector was co-injected, and areas of Gfp expression were separated from the remainder of the organ culture. The expression vectors used are shown at the bottom of each column. Increased Vegfa mRNA is observed in response to injection of a Wt1 expression vector. All Vegfa Ct values were normalized to the Gapdh Ct value from real-time PCR for the injected tissue.

View larger version (47K): [in a new window]   Fig. 3. (A) Vegfa expression in early kidney development compared to Wt1. (a,b) E12.5 kidneys, fixed immediately after dissection and sectioned. (c,d) E11.5 metanephric kidney rudiments placed in culture for 48 hours. (a,c) Vegfa antisense probe, (b) Wt1 antisense probe and (d) Vegfa sense probe. Clear Vegfa expression is present in the condensed mesenchyme of the E12.5 kidney (a) overlapping significantly with Wt1 expression at the same timepoint (b). (B) Vegfa stimulation of branching and tubulogenesis. (a) Control PBS addition, (b) Vegfa addition, Cytokeratin stain red, proximal tubule-binding lectin green. (c) Terminal branch tip (P<0.001) and (d) proximal tubule (P<0.001) quantitation showing results of four pairs of organ cultures represented by (a) and (b), which are from the same embryo. (C) Antibody inhibition of branching. (a) Control organ culture treated with rabbit IgG. (b) Organ culture from the same embryo as (a) treated with anti-Vegfa neutralizing antibody. (c,d) Quantitation of four pairs of organ cultures treated as in (a) and (b); (c) terminal branch tips (P<0.001); (d) tubules as identified by lectin staining for proximal tubules (P<0.001).

View larger version (58K): [in a new window]   Fig. 4. (A) Antibody staining of Flk1, Pax2 and Pecam in E12.5 kidney frozen sections (a-c, g-i) and whole mounts of E11.5 kidney organ cultures placed in culture for 48 hours (d-f, j-l). (a,d) Flk1; (b,e) Pax2; (c,f) merge of preceding panels showing non-overlapping expression of Flk1 and Pax2. (g,j) Flk1; (h,k) Pecam; (i,j) merge of preceding two panels showing Pecam-expressing cells are a subset of Flk1-expressing cells. (B) Flk1-blocking antibody effect on branching and nephrogenesis. Cytokeratin green; proximal tubule brush border antigen red. (a,b) From the same embryo. (a) Treatment with rat IgG; (b) treatment with anti-Flk1; (c,d) quantitation of four pairs of organ cultures represented by (a) and (b), (P<0.001 for both terminal branch number and tubules).

View larger version (62K): [in a new window]   Fig. 5. Flk1 dependency of gene expression. (A,C,E,G,I) treatment with Rat IgG; (B,D,F,H,J) treatment with Flk1 blocking antibody. The probe used for in situ hybridization is shown to the left of each pair of panels. Each pair of panels is representative of the number of experiments noted in Table 1. Similar results were obtained with SU1498, (not shown). (K,L) Real-time PCR for Pax2 (K) and Gdnf (L) demonstrated decreased Pax2 and Gdnf mRNA (P<0.001) after treatment of organ cultures with the Flk1 blocking antibody. All Pax2 and Gdnf Ct values were normalized to Gapdh Ct values and then converted to relative mRNA levels using standard curves, as described in the Materials and methods.

View larger version (46K): [in a new window]   Fig. 6. (A) Flk1-dependent Vegfa induction of Pax2. The injection is noted at the top of each panel, and the antibody treatment and hybridization probes are noted at the bottom of each panel. Gfp (a) or Vegfa (b-e) expression vectors were injected at the edge of the condensed mesenchyme. Rat IgG (d) or Flk1-blocking antibody (e) were added at the time of injection. Six hours after injection, cultures were analyzed by in situ hybridization with Pax2 antisense (a,b,d,e) or sense (c) probe. (B) Flk1-dependent Pax2 induction of Gdnf expression. Panels are labeled as in A. Pax2 expression (a,b.d,f) or control (c,d,g,h) vectors were injected and Gdnf antisense probe was used in all panels. (C) Real-time PCR analysis of Pax2 mRNA expression level in organ cultures after Flk1 blockade or treatment with actinomycin-D. Treatments are shown in the figure box. SU1498, actinomycin-D, SU1498 with actinomycin-D or DMSO, were added to the culture medium at the beginning of the culture period, (time 0). At 0, 1.5, 3, 4.5, 6, or 7.5 hours after beginning cultures, real-time PCR was performed to measure levels of Pax2 mRNA. All Pax2 Ct values were normalized to the Gapdh Ct values and converted to relative mRNA levels as described in the methods. Relative RNA levels are shown on a log scale. (D) Flk1 dependency of Pax2 expression. Panels a-h were injected with the Pax2 expression vector. The hybridization probes are noted at the bottom of each panel (a-d), and the antibody treatment is noted at the top (a-d) or bottom (e-h) of each panel. (a,e) Rat IgG; (b,f) Flk1-blocking antibody; (c,g) DMSO 1:1000; (d,h) SU1498 treatment. (a-d) In situ hybridization with Pax2 antisense probe; (e-h) staining with anti-Pax2 antibody. Pax2 mRNA or protein expression in response to injection of the Pax2 expression vector could be blocked by antibody or pharmacological inhibition of signaling through Flk1. (i-l) Injections of the GFP expression vector, detecting direct GFP fluorescence.

View larger version (45K): [in a new window]   Fig. 7. (A) Gdnf rescue of Flk1 blockade. (a) Flk1 block and (b) Flk1 block with addition of Gdnf are stained for cytokeratin (red) and proximal tubule-binding lectin (green). (a,b) From the same embryo. (c) Terminal branch number (P<0.001) and (d) tubule number (P<0.001) quantification from four pairs of cultures represented by (a) and (b). (B) Pax2 in situ hybridization of organ cultures from E11.5 embryos cultured for 48 hours in the presence of (a) Flk1 block and (b) Flk1 block with addition of Gdnf. (C) Late addition of Flk1 blockade. (a,b) Organ cultures from the same embryo, obtained from E11.5 embryos, cultured for 48 hours before addition of Flk1-blocking antibody (b) or control IgG (a), and cultured for an additional 48 hours. (c,d) Quantitation of four pairs of organ cultures represented by (a) and (b). Differences are not significant.

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