DEVELOPMENT 2397 RESEARCH ARTICLE INTRODUCTION Mammalian kidney organogenesis is regulated by reciprocal epithelial-mesenchymal (e-m) signalling interactions that involve inductive signalling between ureteric bud epithelium and metanephric mesenchyme (Saxén, 1987; Yu et al., 2004). In mouse embryos, the ureteric bud forms around embryonic day (E) 10.5 as an epithelial swelling in the caudal-most part of the Wolffian duct. Development of the definitive kidney is initiated when the ureteric bud elongates to invade and induce the metanephric mesenchyme. In turn, the induced metanephric mesenchyme produces signals that initiate nephrogenesis. During ureteric bud outgrowth, the epithelial tip region thickens to form an ampulla just prior to appearance of the first epithelial branch (Shakya et al., 2005). In addition, this process induces condensation of the metanephric mesenchyme, which is the first morphological sign of nephrogenesis (Saxén, 1987). Many of the human congenital anomalies of the kidney and urinary tract (CAKUT) are caused by defects in these early inductive events, but their aetiology remains poorly understood (Batourina et al., 2002; Pope et al., 1999). During the last decade, significant parts of the molecular networks and e-m feedback signalling interactions that regulate mammalian kidney organogenesis have been identified (Costantini, 2006; Vainio and Lin, 2002). It is established that ureteric bud formation and the induction of its branching require GDNF (glial cell line derived neurotrophic factor), a secreted growth factor that is expressed by the metanephric mesenchyme. The GDNF ligand interacts with its cognate receptor RET (ret proto-oncogene), which is first expressed by the Wolffian duct and then by the ureteric epithelial tips as branching morphogenesis progresses (Schuchardt et al., 1996). The current view is that establishment of signalling between GDNF, RET and its co-receptor GFR1 (glial cell line derived neurotrophic factor family receptor 1) is essential for ureteric bud formation and initiation of outgrowth and branching (Costantini and Shakya, 2006). As the rostral part of the Wolffian duct initially expresses Ret, it is competent to form supernumerary buds and branches upon exposure to GDNF (Shakya et al., 2005). Therefore, a mechanism must exist that restricts ureteric bud formation to the caudal-most part of the Wolffian duct. Indeed, supernumerary epithelial buds form in mouse embryos lacking either SLIT2 or ROBO2 functions, the SPRY1 intra-cellular antagonist or the FOXC1 transcriptional regulator. Molecular analysis showed that SLIT2 and/or ROBO2 signalling is required to restrict Gdnf expression to caudal mesenchyme (Grieshammer et al., 2004). FOXC1 is also required for caudal restriction of Gdnf (Kume et al., 2000), but is not a target of SLIT2/ROBO2 signalling (Grieshammer et al., 2004). By contrast, SPRY1, an intra-cellular antagonist of tyrosine kinase receptors, reduces the sensitivity of the Wolffian duct to GDNF, such that only one ureteric bud forms (Basson et al., 2005; Chi et al., 2004). In Spry1-deficient mouse embryos, ectopic epithelial buds form and multiple- and hydro- 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-Rios 1 , Eva Tiecke 1 , Florence Naillat 2 , Konstantin Beier 3 , Antonella Galli 1 , Seppo Vainio 2 and Rolf Zeller 1,‡ Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling. KEY WORDS: Antagonist, BMP, gremlin 1, Kidney, Mouse, Signalling Development 134, 2397-2405 (2007) doi:10.1242/dev.02861 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. *These authors contributed equally to this work † Present address: Department of Genetics and Development, Columbia University, HHSC-1416, 701 West 168 Street, New York, NY 10032, USA ‡ Author for correspondence (e-mail: Rolf.Zeller@unibas.ch) Accepted 5 April 2007