DEVELOPMENT 151 RESEARCH ARTICLE INTRODUCTION Mammalian adult kidney, metanephros, is formed by reciprocally inductive interaction between two precursor tissues derived from the intermediate mesoderm, the metanephric mesenchyme and the ureteric bud. The ureteric bud induces the metanephric mesenchyme to differentiate into the epithelia of glomeruli and renal tubules, endothelial and stromal cells (Saxen, 1987). Inductive signals have been vigorously investigated, and several factors have been elucidated that trigger epithelialization of metanephric mesenchyme in explant culture system; the members of Wnt family (Herzlinger et al., 1994; Kispert et al., 1998), leukemia inhibitory factor (LIF) (Barasch et al., 1999; Plisov et al., 2001), and transforming growth factor 2 (TGF2) (Plisov et al., 2001). These studies have also suggested the presence of clonal cells in mesenchymal rudiments, which sequentially form renal condensation, comma (C)- and S-shaped bodies, and terminally epithelia of glomeruli and renal tubules, and the existence of single epithelial precursors responding to LIF was demonstrated in mesenchyme (Barasch et al., 1999). One previous report suggested retrospectively the presence of multipotent cells in embryonic kidneys, demonstrating that cells in several portions of nephron were derived from a single stem cell using lacZ gene transduction with retrovirus into a single cell of mesenchyme (Herzlinger et al., 1992). However, none has isolated prospectively the renal progenitor cells with a multilineage differentiation potential from the embryonic kidney, and none has examined their differentiation mechanisms in a single cell culture. There has been a lack of assay systems that specifically identify renal progenitors, as in cases of the neurosphere method for neural stem cells (Reynolds et al., 1992) and the colony assay for hematopoietic progenitors (Pluznik and Sachs, 1965; Bradley and Metcalf, 1966). We previously generated mice in which the green fluorescence protein gene (GFP) was knocked into the locus of Sall1 (Sall1-GFP mice), a zinc finger nuclear factor that is expressed in the metanephric mesenchyme and that is essential for kidney development (Nishinakamura et al., 2001; Takasato et al., 2004). Sall1 is also expressed in the subventricular zone of the central nervous system and progress zones of limb buds, where neural and mesenchymal stem cells reside, respectively, leading to speculation that Sall1 might have some association with stem cells in several organs, including the kidney. Targeted disruption of Wnt4 results in kidney agenesis and impairs mesenchymal-to-epithelial transformation (Stark et al., 1994), and co-culture with 3T3Wnt4 induces tubulogenesis in the mesenchyme rudiment in organ culture (Kispert et al., 1998), suggesting both essential and sufficient roles of Wnt4 for epithelial differentiation of metanephric mesenchyme. Recently, Wnt9b expressed in the ureteric bud was shown to function upstream of Wnt4 (Carroll et al., 2005). Thus, we attempted to set up assay systems that can identify and characterize the progenitor cells with multipotent differentiation potential from uninduced metanephric mesenchyme using Wnt4 signal. Wnt genes are known to regulate multiple cellular functions using at least three intracellular signaling branches: the -catenin pathway (canonical pathway), in which stabilized -catenin interacts with members of the lymphoid enhancer factor/T cell factor (LEF/TCF) family of transcription factors and activates gene expression in the nucleus (Wodarz and Nusse, 1998; Miller et al., 1999); the planar cell polarity (PCP) pathway, which involves Jun N-terminal kinase (JNK) and the Rho family of small guanosine triphosphatases (GTPases) and which directs cytoskeletal rearrangements, coordinated polarization within the plane of epithelial sheets, and morphogenetic movements during development (Veeman et al., 2003; Wallingford et al., 2002); and the Wnt/Ca 2+ pathway, which leads to release of intracellular calcium and is implicated in Xenopus ventralization and in the Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay Kenji Osafune 1,2,3 , Minoru Takasato 2,4 , Andreas Kispert 5 , Makoto Asashima 2,3 and Ryuichi Nishinakamura 1,4,6, * Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP high cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation. KEY WORDS: Progenitor, Kidney, Colony-forming assay, Sall1, Wnt, PCP, JNK, Rho, Mouse Development 133, 151-161 doi:10.1242/dev.02174 1 Division of Stem Cell Regulation, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. 2 Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902, Japan. 3 ICORP, JST, Saitama 332-0012, Japan. 4 Division of Integrative Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860- 0811, Japan. 5 Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany. 6 PRESTO, JST, Saitama 332-0012, Japan. *Author for correspondence (e-mail: ryuichi@kaiju.medic.kumamoto-u.ac.jp) Accepted 24 October 2005