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First published online 16 June 2004
doi: 10.1242/dev.01200

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Sprouty proteins regulate ureteric branching by coordinating reciprocal epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney development

Lijun Chi^*, Shaobing Zhang^*,, Yanfeng Lin, Renata Prunskaite-Hyyryläinen, Reetta Vuolteenaho, Petri Itäranta and Seppo Vainio^¶

Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland

View larger version (23K): [in a new window]   Fig. 1. Targeted expression of human SPRY2 and EGFP in the ureteric bud in vivo. (A) Schematic structure of the expression construct generated by inserting a PCR fragment of human SPRY2 cDNA into the EcoRI and BamHI sites of a plasmid containing IRES-EGFP. The fragment was excised with EcoRI and SspI, and inserted into the EcoRI and SmaI sites of a modified bluescript vector. A ß-globin splice acceptor was then cut with BamHI and EcoRV, blunted and inserted into a blunted NotI site (N) downstream of the Pax2 promoter. The fragment containing the Pax2 promotor and ß-globin was removed from the vector with EcoRI and ligated upstream of the human SPRY2 cDNA, IRES EGFP-PA. The yellow arrow indicates the start site of transcription. B, BamHI; EcoRI site, (N) defective NotI site; E, EcoRI. (B) An expected 300 bp fragment was amplified with P1 and P2 primers in PCR, indicating the presence of the transgene in the genome of the carriers. (C) The copy number of the transgene was estimated by Southern blotting using GFP as a probe. Control of loading is indicated below. The Pax2 promoter drives expression of EGFP in the ureteric bud of a kidney at E11.5 (D) and E17.5 (F). The human SPRY2 gene is also expressed in the kidney at E12.5 (E) and E17.5 (G), as judged by in situ hybridization. WT, wild type; TG, transgene carrier. Scale bars: 100 µm.

View larger version (80K): [in a new window]   Fig. 2. Human SPRY2 expression leads to severe defects in kidney development. When compared with the kidney of a wild-type embryo (A,B), human SPRY2 (TG) expression leads to reduced size of the organ (C,D; E17.5), unilateral agenesis (F, arrow on the right), cystogenesis with a blind-ended hydroureter (F,G,K; F,G, arrows), unilateral lobularization of the kidney (H, stars; E17.5) with cysts (C1-C3 in K; E17.5) or an ectopic second ureteric bud (K1 and K2 in L) when compared with wild-type (WT) controls (A,B,E,I; E17.5). Reduction in the size of the kidney is associated with reduced cell proliferation (M; ***P<0.005) between the wild-type and transgenic organ in derivatives of the ureteric bud and glomeruli (N,O arrows; E15.5). Human SPRY2 expression also leads to stimulation of apoptosis (P; ***P<0.005) in derivatives of the ureteric bud and glomeruli (Q,R arrows). Scale bars: 100 µm. K1, host kidney; K2, ectopic kidney; C1-C3, cystic lobules).

View larger version (124K): [in a new window]   Fig. 3. Expression of candidate targets for human SPRY2-mediated antagonism of Fgf genes and Fgfr genes in the embryonic kidney. Fgf2 is expressed in the ureteric bud, mesenchymal cells and assembling nephrons, and human SPRY2 expression leads to reduced Fgf2 gene expression in the ureteric bud (compare A with A',A''). Expression of Fgf7, a stromal marker (Finch et al., 1995), is also reduced because of human SPRY2 expression when compared with the normal kidney (compare B with B',B''), while Fgf8 remains unchanged (C,C',C''). Fgf9 is again reduced, especially when the transgene is inherited from both the female and male (compare D,D' with D''). Fgfr1, which is expressed in mesenchymal cells and in developing nephrons (E arrows), is not detected in more mature nephrons, owing to human SPRY2 expression (compare E,E' with E''). Expression of Fgfr2, Fgfr3 and Fgfr4 appeared to be unchanged in the presence of human SPRY2 gene expression (F-H''). (A-H'') E15.5. Scale bars: 100 µm. Fgfr, Fgf receptor; WT, wild type; TG, explants containing human SPRY2.

View larger version (87K): [in a new window]   Fig. 4. Ectopic human SPRY2 in the ureteric bud leads to deregulated expression of Wnt11/Gdnf and Foxd1 and is associated with reduced branching. (A) Gdnf is expressed in the kidney mesenchyme, its gene encoding the Ret receptor and another gene Wnt11 in the ureteric bud (B,C). At the same developmental stage, Gdnf (A') and Wnt11 (C') expression is reduced in response to human SPRY2 expression, while Ret remains unchanged (B'). Note that there are less ureteric tips in the kidneys expressing human SPRY2, as indicated by the tip markers (compare B,C with B',C'). While Bmp4 expression shows no notable changes (D,D'), the stromal marker Foxd1 is clearly reduced due to human SPRY2 expression (compare E with E'). Mouse Spry1 expression remains constant irrespective of expression of the human SPRY2 transgene (F,F'). (A-F') E12.5. Scale bars: 100 µm. WT, wild type; TG, explants containing human SPRY2.

View larger version (102K): [in a new window]   Fig. 5. Expression of mesenchymal, epithelial and stromal markers in the embryonic kidney expressing human SPRY2. Gdnf, which is expressed in the nephrogenic mesenchyme, is downregulated by human SPRY2 (compare A with A' and A''), while its Ret receptor encoding gene remains normally expressed irrespective of the presence of human SPRY2 (B-B''). Wnt11 expression is reduced in response to human SPRY2 expression (compare C with C',C''). Wnt11 also appears in the stromal cells of the transgenic kidney (C',C''). Pax2 and Bmp4 gene expression is unchanged (D-E''), while the stromal marker Foxd1 is reduced (F-F'') because of ectopic human SPRY2 (A-F''; E.15.5). Scale bar: 100 µm.

View larger version (87K): [in a new window]   Fig. 6. Human SPRY2 mediates its effect via Fgf genes and resembles the phenotype induced by inhibition of the ERK/MAPK pathway. Although local application of an agarose bead soaked with BSA, Fgf8 or Fgf10 (A,C,D) does not alter Spry2 gene expression, a Fgf7-soaked bead (B) induces its expression in culture when compared with the contralateral side that serves as a control (arrow). The asterisk in D indicates the site of the bead. (E) The kidney of a normal E11.5 embryo cultured for 48 hours shows an extensively branched ureteric bud (indicated in orange) and induced nephrons (indicated in green). (F) The kidney from an embryo expressing human SPRY2 has a ureteric bud with less branching and no nephrons at this stage of culture. (G) Quantification of the reduction in the degree of ureteric branching between wild-type and human SPRY2 kidneys (***P<0.005). A 48 hour culture. (H,K) Fgf7 and Gdnf, when administered to the culture media, alone (H) or in combination (K; Fgf2,7,10), lead to substantial recovery in the branching defect affecting the ureteric buds of the kidneys expressing human SPRY2 (compare H,I,K,L with F), whereas Fgf2 does not (J). Fgf7 signalling (H), a combination of Fgf7, Fgf2 and Fgf10 signalling (K), and GDNF signalling (L) induce the formation of supernumerary epithelial buds from the Wolffian duct (arrows), indicating that human SPRY2 sensitizes the kidney to these signals. (M) Quantification of the responses of human SPRY2 transgenic kidneys. Significant differences (**P<0.05), numbers of samples and growth factors analysed are indicated. Administration of PD98059, an inhibitor of ERK/MAP kinase, reduces branching of the ureteric bud in a normal kidney (compare N,O with E) and has an additive influence on the reduction in ureteric elongation when human SPRY2 is expressed (compare P,Q with F). Scale bar: 100 µm.

View larger version (89K): [in a new window]   Fig. 7. Schematic model of some mediators of inductive tissue interactions in the embryonic kidney. Ret, a tyrosine kinase receptor, and its ligand Gdnf are essential for kidney development (Airaksinen and Saarma, 2002). Wnt11 is necessary for Gdnf expression, while Ret signalling regulates Wnt11 expression (Majumdar et al., 2003). Stromal components in the retinoic acid (RA) signalling system also contribute to Ret expression (Batourina et al., 2001), perhaps indirectly. Sprouty signalling may contribute to the coordination of Wnt11/Gdnf/Ret pathways (Majumdar et al., 2003). As a RTK receptor effector, Sprouty may either directly antagonize Ret function or have an indirect effect via changes in reciprocal stromal signalling, such as Fgf7 or Foxd1-regulated signals (Hatini et al., 1996). Fgf7 signalling regulates Spry2 expression and is also influenced by human SPRY2 expression in the ureteric bud (red arrow), suggesting a link in inductive signalling between epithelial ureteric bud (U) and stromal cells (S). W, Wolffian duct; N, nephrogenic mesenchyme.