Morphogenesis in the differentiating metanephros is regulated by reciprocal interactions between ureteric bud (UB) epithelia and the metanephric mesenchyme (MM). The UB invades the overlying MM and induces conversion of the mesenchyme into stromal and epithelial elements, which form the nephron. In turn, the MM stimulates the UB to grow and branch, forming the collecting duct system. The Differentiation and Neoplasia Section has focused on the elucidation of mechanisms of inductive signaling in metanephric development, seeking (1) the ligands responsible for renal progenitor survival and nephronic differentiation, (2) other non-inductive regulatory factors of nephrogenesis, (3) the molecular targets of induction, and (4) alterations in normal inductive signaling during tumorigenesis. Thus far, we have identified three families of secreted soluble growth factors/cytokines produced by the UB that cooperate in inductive signaling to generate nephronic tubules. These include the fibroblast growth factors (notably FGF2 and 9), gp130-binding proteins (leukemia inhibitory factor; LIF), and transforming growth factor-beta (Tgf-beta2, activin A and B, and gdf11). Through gene targeting studies, we have additionally identified Fgf8, a factor expressed by MM, as an essential player in nephrogenesis through its upstream effects on expression of the secreted patterning molecule Wnt4 and homeodomain transcription factor Lim1. We have determined that both Fgf8 and a Wnt are required to rescue kidney development, suggesting that they may cooperate in inducing Lim1 for tubule formation. Indeed both Wnt4 and Fgf8 are capable of regulating Lim1 expression. In the genetic studies, we have also discovered a role for Fgf8 in the development of male reproductive tract accessory tissues. The phenotype resembles that caused by the loss of Lim1 expression in the Wolffian duct/UB, not the nephrogenic cord/MM. Since these tissues are believed to arise from the Wolffian duct, this may suggest that Fgf8 also acts on the UB. We are currently evaluating this phenotype. To understand the targets of individual signaling pathways and their cooperative output, we have begun a systematic assessment of activated inductive signaling in the MM. As part of this effort, we have generated an inducible immortalized cell line of MM that, under inductive conditions, differentiates into tubular epithelia and responds to the three described families of inductive factors. Furthermore, microarray analysis has provided a comprehensive characterization of transcriptional targets associated with induction. We are using this line along with primary MM to characterize the proteomic changes that mediate or regulate differentiation of the MM progenitor. Since Wnt4 has been shown to be essential for the epithelial conversion of MM and since a TCF-dependent canonical Wnt reporter is not active in the MM during mouse development, Wnt4 may function through an alternative or noncanonical Wnt pathway. Using a series of chemical inhibitors for components in canonical or noncanonical pathways, we have determined that signaling through more than one Wnt pathway contributes to tubule formation. A GSK-3beta inhibitor BIO proved to be a potent inducer of MM progenitor survival and tubulogenesis and may prove useful in distinguishing the cytoplasmic versus nuclear roles of beta-catenin in differentiation. The mechanism for its action has not been established in our test system; however, we have determined using a kidney cell line that it increases beta-catenin levels (consistent with its effects on GSK-3beta) and stimulates TCF activation/canonical signaling. In addition, a Rho kinase inhibitor Y27632 blocks tubule formation in MM, suggesting that the noncanonical Rac/Rho-mediated Wnt pathway also plays a role. We are currently confirming its effects on this pathway using levels which block tubulogenesis.
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