Part B: Signalling in Polycystic Kidney Disease: Wnt signalling in kidney development and disease
Final Report Abstract
The overall goal of this project concerned the elucidation of the role of the canonical Wnt signaling pathway in kidney development. We analyzed the kidney development in homozygous Lef1-β-gal mice, and we could confirm the initial observation that these mutant mice lack kidneys. The analysis of the mutant mice was performed at birth as the mice die shortly after birth. However, we also did not detect any rudiments of a developing kidney in earlier stage embryos, suggesting that kidney development is blocked a very early stage. To overcome the problem of apparent kidney agenesis, we attempted to generate a hypomorphic strain by introducing the Tcf1V hypomorphic allele into the Lef1-β-gal background. Unfortunately, this allele is no longer available from Dr. H. Clevers (Utrecht) who generated the mutant mouse. We have now the Tcf1VII null allele, and we will use this null allele for crossing with Lef1-β-gal mice. Another goal of the project was the use of various mouse models to study Wnt-mediated processes in kidney development. Based on the delay in the generation of a conditional allele of Lef1, we decided to use other mouse models in the meantime. First, we examined a mouse in which point mutations in the β-catenin interaction domain abrogate the Wntdependent, but not Wnt-independent functions of Lef1. These mice have no apparent kidney defect and are now being crossed onto the Tcf1VII null background. Second, we explored the role of Bcl9, an adaptor protein that couples Pygopus protein to β-catenin. We have previously shown that Bcl9 acts in a cell type-specific manner to augment the nuclear Wnt response. To gain insight into the function of Bcl9 in the transcriptional response to Wnt signaling, we performed an siRNA screen in Drosophila S2 cells, using the carboxyl-terminal transactivation domain of Bcl9, which synergizes with the transactivation domain of β-catenin. In this assay, we identified ~263 genes from which twenty genes could be confirmed in a secondary screen. We are now performing coimmunoprecipitations and siRNA knock-down experiments in mammalian cells to further examine the potential function of these genes in Bcl9-mediated Wnt signaling. Based on the reported kidney defects in Pygopus mutant mice, we also obtained Bcl9- and Bcl9L-floxed mice from Michel Aguet (Lausanne). Bcl9 and Bcl9L act in a redundant manner and therefore, we are currently examining Bcl9- and Bcl9L-double deficient mice for kidney defects.
