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doi: 10.1242/10.1242/dev.00520

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Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development

¹ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
² Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, FIN-90014, Oulu, Finland
³ Institut für Molekularbiologie, OE5250, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

View larger version (45K): [in a new window]   Fig. 1. Gene targeting of the murine Wnt11 locus. (A) Targeting strategy. Genomic sequences spanning the Wnt11 locus were cloned and subjected to restriction mapping and sequencing to locate the intron-exon boundaries. The homologous recombination events lead to deletion of exons 4 and 5 and around 1.5 kb of intron 5 and lead to generation of a truncated transcript at amino acid 28 onwards in the corresponding Wnt11 protein. The 5', 3'and neo probes used to screen for gene targeting with Southern blot are indicated, and SpeI digestion was used as a diagnostic enzyme to screen the targeting event. Cutting of the wild-type locus with SpeI was expected to generate around a 20 kb fragment where the PGKneo introduces additional SpeI in the targeted allele and was expected to generate a 10 kb fragment. Primers to monitor the wild-type and mutant allele are also indicated with arrowheads. (B) Genotyping the Wnt11 knock out allele. Genomic DNAs from Wnt11 wild-type, heterozygote and homozygous mutant alleles were digested with AflII, Southern blotted and probed with the Wnt11 cDNA 240 bp kb Nco fragment within Exon VI. The Wnt11 allele specific polymorphism is shown whereby the Wnt11 wild-type allele is associated with a 7.0 kb band, while the Wnt11 mutant allele is associated with a 5.5 kb band. (C) Wnt11 homozygous mutant kidneys produce a shortened Wnt11 mRNA. Genespecific primers were used to RT-PCR wild-type and mutant P1 kidney mRNA. The wild-type product is 1.8 kb, while the Wnt11 mutant product is 1.3 kb, in agreement with the expected size resulting from deletion of Wnt11 exons IV and V. (D) The first 28 amino acids of the mutant Wnt11 protein match the wild-type sequence. Downstream of the exon IV/V deletion (arrow), the reading frame is out of frame resulting in a null allele.

View larger version (98K): [in a new window]   Fig. 2. Wnt11–/– mutants have smaller kidneys. Comparison of urogenital systems from wild-type (A) and Wnt11–/– mutant (B) P1 pups shows Wnt11–/– mutants have reduced kidney size. Hematoxylin/Eosin staining of coronal 6 µm sections from wild-type (C) and Wnt11–/– mutant (D) kidneys is shown. In Wnt11–/– kidneys, gross cortico-medullary patterning and epithelial integrity appears normal and the ureteric epithelium has undergone extensive branching. Sections are made at the level of the pelvis. Scale bars: 1 mm in A,B; 500 µm in C,D.

View larger version (142K): [in a new window]   Fig. 3. Timecourse of ureteric branching during wild-type development. Wild-type left (A,A',B,B',C,C') and right (D,D',E,E',F,F') E12.0, E12.25 and E12.5 kidneys were stained with Ret antisense in situ probe. Ret is expressed specifically in the ureteric epithelium and is used here to visualize the collecting duct system. Ret is expressed at higher levels in forming ureteric tips and at lower levels in stems. In A, arrow indicates the Wolffian duct. Ret staining at these stages visualizes the trifurcation event that gives rise to new ureteric tips. At E12.0 (A,D), Ret is expressed in the two ampullae at the T stage. Ret expression appears pronounced in cells at the vertices of the triangle-shaped ampullae (arrowheads). At E12.5 (B,E), Ret is expressed most strongly at each of the three vertices of the emerging in the trifurcation. By E12.5 (C,F), Ret remains strongly expressed in the new morphologically distinct tips. In 42% of kidneys, a seventh ureteric tip emerges (arrowheads in insets A'-F',I') and undergoes morphogenesis to become a distinct tip. The entire branching ureteric epithelium is visualized in kidneys from Hoxb7 Cre; Rosa26 YFP embryos (G-I,I'). Arrows indicate the Wolffian duct. Ureteric specific YFP expression visualizes the emerging tips of the trifurcation and their morphogenesis into distinct tips. In I', an example is show where a seventh tip emerges from the original point of bifurcation during the T stage (arrowhead). In addition, the metanephric kidney has undergone a rotation during these stages such that the mediolateral axis present at E11.5 has translated to a dorsoventral axis by E12.5.Both left and right kidneys show similar patterns of branching. Double-headed arrows in A and B indicate orientation [anterior (A), posterior (P), dorsal (D) and ventral (V)]. Scale bars: 100 µm.

View larger version (102K): [in a new window]   Fig. 4. Branching defects occur during the trifurcation event in Wnt11–/– kidneys. Wild-type (A,C,E,G,I,K,M,O) and Wnt11–/– mutant (B,D,F,H,J,L,N,P) kidneys at E11.5 (A-D), E12.0 (E-H), E12.25 (I-L) and E12.5 (M-P) were stained with Ret (A,B,E,F,I,J,M,N) or Wnt11 (C,D,G,H,K,L,O,P) antisense in situ probes to visualize the branching process. By E11.5, ureteric bud invasion of mesenchyme and one round of branching have occurred in wild-type to generate the T stage. Ureteric invasion of mesenchyme occurs on schedule in Wnt11–/– kidneys (C,D). In wild type, both Ret and Wnt11 are expressed strongly in the ureteric tips emerging from the trifurcation (E,I,G,K). In Wnt11–/– kidneys, Ret is expressed in the ureteric epithelium; however, the branching process appears retarded. E12.0 and E12.25 Wnt11–/– kidneys appear to be lagging behind in making new ureteric tips. In wild type E12.5 kidneys, Ret and Wnt11 are expressed strongly in the new ureteric tips (M,O). In Wnt11–/– E12.5 kidneys, Ret and Wnt11 expression patterns indicate loss of tips (compare N with M and P with O). For all these experiments, kidneys are taken from same stage embryos with identical lung branching pattern (see Materials and Methods). Kidneys are oriented anterior towards the top and posterior towards the bottom.

View larger version (53K): [in a new window]   Fig. 5. Wnt11 and Ret/Gdnf signals are mutually dependent. Gdnf is downregulated in Wnt11–/– kidneys. Gdnf expression in E12.5 wild-type (A) kidneys is found in the mesenchyme surrounding the non-staining ureteric epithelium. Mesenchymal Gdnf expression is reduced in E12.5 Wnt11–/– kidneys (B). By contrast, Pax2 continues to be expressed in Wnt11–/– kidney mesenchyme at E12.5 (compare D with C). Wnt11 expression is reduced in Ret–/– kidneys. Wnt11 expression in wild-type (E) E12.0 kidneys marks the forming ureteric tips during the trifurcation stage. Wnt11 expression is dramatically reduced in E12.0 Ret–/– kidneys (arrows in F). However, Emx2 continues to be expressed in Ret–/– ureteric epithelium comparable with wild type (compare H with G). Kidneys are oriented anterior towards the top and posterior towards the bottom.

View larger version (111K): [in a new window]   Fig. 6. Synergystic genetic interactions between Wnt11 and Ret. Wild-type (A,E,I), Ret+/–; Wnt11+/– (B,F,J), Wnt11–/– (C,G,K) and Wnt11–/–; Ret+/– (D,H,L) kidneys are shown. (A-D) E18.5 urogenital systems. (E-H) Hematoxylin/Eosin staining of E18.5 kidney sections taken at the level of the pelvis. (I-L) E12.5 kidneys in situ hybridized to a Ret probe. Wnt11+/–; Ret+/– kidneys are smaller than wild type (compare F with E), despite having a normal ureteric branching pattern at E12.5 (compare J with I). Wnt11–/– Ret+/– kidneys are smaller than wild type, Wnt11–/– or Wnt11+/–; Ret+/– kidneys (compare H with E-G) and show much more severe branching defects at E12.5 (compare L with I-K). Ret+/– E12.5 kidneys appear wild type in size and in their branching pattern. In I-L, kidneys are oriented anterior towards the top and posterior towards the bottom, dorsal towards the left and ventral towards the right. Scale bars: in A, 1mm for A-D; in E, 500 µm for E-H; in I, 100 µm for I-L.