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First published online 23 May 2007
doi: 10.1242/dev.02861

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Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Odyssé Michos^1,*,, Alexandre Gonçalves^1,*, Javier Lopez-Rios¹, Eva Tiecke¹, Florence Naillat², Konstantin Beier³, Antonella Galli¹, Seppo Vainio² and Rolf Zeller^1,

¹ Developmental Genetics, DKBW Centre for Biomedicine, University of Basel Medical Faculty, Mattenstrasse 28, CH-4058 Basel, Switzerland.
² Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu, Laboratory of Developmental Biology, Aapistie 5A, PO Box 5000, University of Oulu, F-90570 Oulu, Finland.
³ Department of Histology, Anatomy Institute, Pestalozzistrasse 20, CH-4056 Basel, Switzerland.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Recombinant GREM1 protein is able to induce ectopic epithelial buds and restore branching in Grem1-deficient kidney primordia. Wild-type and Grem1-deficient kidney primordia expressing the Hoxb7-GFP transgene in their Wolffian duct and ureteric epithelium (Srinivas et al., 1999) were isolated from mouse embryos of 40-44 somites (E11.0-E11.25) and cultured for up to 96 hours. Panels show from left to right: cultures at 0 hours, 24 hours, 48 hours, 72 hours and 96 hours (time: ±2-3 hours). White asterisks indicate ureteric buds, red asterisks indicate ectopic epithelial buds and blue asterisks ectopic branches. The right-most panels are schematic tracings of the Wolffian duct and epithelial branching pattern at the developmental time points indicated (96 hours for Grem1 mutants; 72 hours for wild type). Wolffian duct and ureteric bud are shown in white; ectopic epithelial buds and/or outgrowth in yellow; first branch in red; second branch in blue; third branch in green; fourth branch in purple; fifth branch (A only) in orange. (A,B) Kidney primordia cultured in control medium. (A) Wild-type control. (B) Grem1-deficient metanephros; outgrowth and branching are blocked. (C-E) Grem1-deficient (C,D) and wild-type (E) kidney primordia cultured in medium supplemented with recombinant GREM1 (5 µg/ml). The Grem1-deficient metanephric kidney primordia shown in C and D are representative of the variability observed.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Treatment with recombinant GREM1 enables upregulation and propagation of Wnt11 and Gdnf expression in Grem1-deficient kidney rudiments in culture. Mouse kidney primordia were isolated at E10.75-11.0 (38-42 somites) and cultured for 48 hours either without (A,B,D,E) or in the presence of recombinant GREM1 (5 µg/ml, C,F). All upper panels show the epithelial branching pattern as revealed by the Hoxb7-GFP transgene. Lower panels show transcript distributions as revealed by whole-mount in situ hybridisation. Ureteric buds are indicted by white asterisks, ectopic epithelial buds by red asterisks. (A-C) Gdnf expression: (A) wild-type control; (B) Grem1-deficient kidney primordia, note the smaller size and remaining Gdnf expression; (C) Grem1-deficient kidney primordia cultured in the presence of recombinant GREM1. Note the ectopic epithelial buds and Gdnf expression in the surrounding mesenchyme. (D-F) Wnt11 expression (D) wild-type control; (E) Grem1-deficient kidney primordia, note the complete loss of Wnt11 expression; (F) Grem1-deficient kidney primordia cultured in the presence of recombinant GREM1. Wnt11 is expressed in the epithelial tips of both ureteric and ectopic buds.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Recombinant GDNF induces epithelial overgrowth and excessive branching in both wild-type and Grem1-deficient mouse metanephric kidney primordia. Kidney primordia (E11.0, 40-42 somites) were cultured for 96 hours in medium supplemented with recombinant GDNF (100 ng/ml). (A) Wild type; note the formation of many epithelial buds along the Wolffian duct and overgrowth of the ureteric epithelium within 24 hours, which results in excessive branching. (B) Grem1-deficient kidney primordia; note the formation of many epithelial buds, overgrowth and excessive branching similar to the wild type (A).

View larger version (61K): [in this window] [in a new window]   Fig. 4. Dynamic changes in Grem1 and Bmp4 expression during initiation of metanephric kidney development in mouse. (A-D) Bmp4 distribution during initiation of metanephric kidney development. (A,B) GFP reveals the morphology of the nascent ureteric bud at E11.0 (40-42 somites; A) and after the first epithelial branching (E11.5, 48-50 somites; B). Asterisks in B indicate the tips of the ureter branches. (C,D) The Bmp4 transcript distribution was determined using a lacZ reporter gene inserted into the endogenous Bmp4 transcription unit (Bmp4:lacZ) in heterozygous embryos (Kulessa and Hogan, 2002), as detection by whole mount in situ hybridisation is not reliable. (C) At E11.0, Bmp4:lacZ is expressed by the mesenchyme surrounding the Wolffian duct (arrow) and ureteric bud (see also G). The dotted circle indicates the position of the ureteric bud (compare with A). (D) By E11.5, the Bmp4:lacZ expression is retained in the mesenchyme surrounding the Wolffian duct (arrow), while the metanephric mesenchyme is largely devoid of Bmp4. The dotted line indicates the position of the first branch (compare with B). (E,F) Grem1 expression in the mesenchyme. (E) At E11.0, Grem1 expression is highest in the mesenchyme around the ureteric bud (indicated by a dotted circle). (F) Subsequently (E11.5), Grem1 is expressed in the mesenchyme around the tips of first ureteric epithelial branch (indicated by dotted oval). (G,H) Detection of Bmp4 and Grem1 transcripts on transverse sections by in situ hybridisation. Sections are oriented with dorsal to the top. Note that the epithelium (indicated by a dotted line) expresses neither Bmp4 (G) nor Grem1 (H). (G) There is abundant Bmp4 expression in the mesenchyme around the caudal Wolffian duct and ureteric epithelium in agreement with the Bmp4:lacZ distribution shown in C. (H) At E10.75 (38 somites), Grem1 is also expressed in the mesenchyme around the caudal Wolffian duct and ureteric epithelium in agreement with the whole mount in situ hybridisation shown in E. Note that Bmp4 and Grem1 are also expressed by the mesenchyme around the hindgut. Hg, hindgut; mm,: metanephric mesenchyme; wd, Wolffian duct.

View larger version (143K): [in this window] [in a new window]   Fig. 5. Detection of phosphorylated SMAD1/5/8 proteins (pSMAD) on tissue sections. The brown precipitate reveals pSMAD-positive nuclei. All sections are counterstained with Hoechst 33258 (light blue fluorescence) to reveal nuclei lacking pSMADs. All sections are sagittal along the primary embryonic axis with ventral to the left and dorsal to the right. Sections shown are representative of the results obtained by analysing serial sections of at least three embryos per genotype. (A,C,E) Wild-type mouse embryos at E11.0 (40 somites; A); E11.25 (47 somites; C) and E11.5 (50 somites, enlargement of tip region; E). (B,D,F) Grem1-deficient embryos at E11.0 (42 somites; B) and E11.25 (48 somites; D), an enlargement of the ureteric bud area in D is shown in F. The area within the red dashed line contains many picnotic nuclei indicative of the onset of cellular apoptosis (D,F). cm, condensing metanephric mesenchyme; mm, metanephric mesenchyme; sm, stromal mesenchyme; ub, ureteric bud; ue, ureteric epithelium (first branch); us, ureteric stalk; wd, Wolffian duct.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Genetic inactivation of one copy of the Bmp4 gene in a Grem1 mutant background rescues the kinetics of ureteric epithelial growth and branching. Left panels: GFP reveals the morphology of the epithelium before fixation. Right panels: whole-mount in situ detection of the expression of the Ret receptor (A-C,G-I) and the Gdnf ligand (D-F,J-L). (A,D,G,J) Wild-type metanephric primordia. (B,E,H,K) Grem1 (G1/)-deficient metanephric primordia. Note the complete disruption of ampulla formation (E,H,K) and loss of mesenchymal Gdnf expression (K), whereas Ret expression remains in the arrested epithelium (H). (C,F,I,L) Metanephric primordia of G1/; B4/+ mouse embryos. Note the rescue of ureteric epithelial outgrowth (C), branching (F,I,L) and propagation of Gdnf expression (L).

View larger version (132K): [in this window] [in a new window]   Fig. 7. Inactivation of one copy of the Bmp4 gene in a Grem1-deficient mouse embryo results in formation of two normal kidneys. (A-C) Whole-mount in situ hybridisation to detect Pax2 expression at E14.5. (A) Wild-type Pax2 distribution. (B) The absence of Pax2 expression in a Grem1-deficient embryo is due to the complete renal aplasia at this stage. (C) The Pax2 distribution in kidneys of a Grem1/; Bmp4/+ embryo is indistinguishable from a wild-type littermate (A). (D) Gross morphology of the wild-type urogenital system at birth (postnatal day P0). (E) Haematoxylin and Eosin staining of a histological section of a wild-type kidney 30 days after birth (P30). (F) PAS staining reveals the morphology of the medulla (collecting ducts) and cortex regions of a wild-type kidney at about 8 months of age (P250). (G) Gross morphology of the urogenital system of a Grem1/; Bmp4/+ mouse at birth (P0). Note that both kidneys are of normal size and shape (compare with D). (H) Haematoxylin and Eosin staining of a kidney section from a Grem1/; Bmp4/+ mouse at P30. (I) PAS staining of a kidney from a Grem1/; Bmp4/+ mouse at P250. Medulla, cortex and glomeruli appear normal. Bl, bladder; cd, collecting duct; cx, cortex; gl, glomerulus; go, gonad; ki, kidney; md, medulla, tu, tubules.

View larger version (38K): [in this window] [in a new window]   Fig. 8. Reduction of BMP4 activity by gremlin 1 in the mesenchyme around the ureteric bud is essential to enable ureteric epithelial outgrowth, GDNF-RET and WNT11-mediated e-m feedback signalling and branching morphogenesis. (A) In mouse, the ureteric bud forms in the caudal-most part of the Wolffian duct under the influence of GDNF-RET signalling. During this inductive period, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct. High levels of mesenchymal BMP4 activity inhibit the formation of ectopic epithelial buds and epithelial branching at this stage (prior to E11.0). At this early stage only low levels of Grem1 transcripts are detected (not shown). (B) Expression of the BMP antagonist Grem1 is upregulated in the mesenchyme around the nascent ureteric bud thereby locally reducing BMP4 signal transduction (around E11.75-11.0). This reduction of BMP4 activity by GREM1 enables initiation of ureteric bud outgrowth and its invasion into the metanephric mesenchyme. (C) GREM1 is required to maintain and propagate expression of Wnt11 in the ureteric epithelial tip(s) and Gdnf in the mesenchyme via e-m feedback signalling. For details see Discussion.