Through mouse gene targeting studies, we have identified Fgf8, a factor expressed by metanephric mesenchyme (MM), as an essential factor 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 these studies, we have also discovered a role for Fgf8 in the development of male sex accessory tissues. During development of the mesonephros, the rostral aspect of the Wolffian duct (WD) (or cranial tubules) is absent in mutant animals, resulting in the loss of the head and body of the epididymis and fragmentation of the vas deferens. Surprisingly, the Mullerian duct appears unaffected despite its close association with WD development, suggesting that this relationship is mediated by caudal elements of the WD. These findings demonstrate for the first time the critical role of Fgf8 signaling in formation of the male accessory reproductive tract tissues.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. Since Wnt4 and Wnt9 are essential for the epithelial conversion of MM, but Wnt-mediated transcriptional activation is not evident using a transgenic reporter, we have assessed the role of the canonical Wnt mediator beta-catenin in cells from MM. A GSK-3beta inhibitor BIO proved to be a potent inducer of MM progenitor survival, proliferation, and tubule formation. We have found that BIO stabilizes beta-catenin and activates Wnt-dependent signaling in cultured cells from MM. Its differentiation-inducing activity is blocked using neutralizing antibodies for members of the cadherin family, which interact with beta-catenin in adhesion complexes in the cell membrane. Furthermore, an inhibitor of the MAP kinase pathway abrogates BIO-induced tubule formation, suggesting that signaling through this pathway also plays a critical role in mesenchymal-epithelial transition in the kidney. To better understand the role of beta-catenin in vivo, we are collaborating with Drs. Mark Lewandoski and Terry Yamaguchi to generate a UB or a MM-specific conditional loss-of-function and gain-of-function transgenic for beta-catenin. Thus far, we have begun an evaluation of the UB-specific transgenics and have found that branching morphogenesis of the UB is significantly affected in both knockout and knockin mice. Therefore, beta-catenin and, presumably, Wnt signaling plays a role in this process. In the pediatric Wilms tumor, transcriptional activation of the canonical Wnt mechanism, i.e., TCF activation, is constitutive in the majority of tumors. In tumor cell lines, we have found that BIO induces two molecular forms of beta-catenin: one which interacts with E-cadherin and the other with TCF. It also promotes the formation of adhesion complexes in tumor cells, facilitating mesenchymal-to-epithelial transition. Moreover, blocking formation of adhesion complexes using a mutant form of cadherin blocks epithelial transition. We are now assessing whether this treatment affects the tumorigenic phenotype of these cells. We predict that increased adhesiveness will inhibit their tumorigenicity, including invasive behavior. The ultimate goal of the DNS is to define inappropriate signaling in tumorigenesis based upon mechanisms required for the differentiation of normal tissue progenitors during development and tissue renewal. Accordingly, having identified multiple pathways that mediate normal cell maintenance and differentiation in the kidney, we have shifted efforts to the Wilms tumor, a caricature of metanephric development which is characterized by an accumulation of blastemal progenitors.
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