Morphogenesis and stem cell fate determination are directed by a complex of soluble factors and extracellular matrix interactions in organogenesis. While many of these factors have been defined, there is no comprehensive understanding of the specific molecules that mediate differentiation in any given tissue, the responses that each factor elicits, or the sequence of events required for proper patterning. Accordingly, we have developed in vitro systems for producing and purifying the inductive factors that direct kidney development and for analyzing molecular responses in renal tissues to this induction. Previously, we established a cell line from the ureteric bud epithelium, the renal inductive tissue and precursor for the collecting duct in the adult. These cells grow in a defined medium and secrete a tubule-inducing/inductive activity for metanephric mesenchyme. The inductive factor(s) produced by these cells can be collected from conditioned medium, and one of these has recently been purified. This protein has been shown to mediate both the early events of nephrogenesis, i.e., condensation of mesenchyme and upregulation of wt1, as well as late events, i.e., tubulogenesis and upregulation of lim-1. A second factor, which accelerates tubulogenesis in combination with the first, is also suggested from the purification. As an extension of this work, we have also initiated efforts to characterize events downstream of inductive signaling and specifically those associated with the epithelial conversion of metanephric mesenchyme. For this, we previously showed that Fgf2 can mediate condensation but not tubule formation in explant culture, while Fgf2 in combination with our bud cell line-derived activity can induce complete nephrogenesis. The differences have provided the basis for a direct evaluation of the genes responsible for morphogenesis using differential display methodologies. Tubulogenesis is completed over a 3-day period, so molecular events were evaluated by processing explants after 6, 24, and 72 hrs. Using this approach, we have identified 72 cDNA species, which are specifically regulated in this process, 36 of which are novel. These studies have identified certain signaling pathways that mediate the inductive response and include growth factors, cell-cycle regulatory proteins, protein kinases involved in signal transduction pathways, calcium-binding proteins, transcription factors, and proteins associated with cell adhesion. A picture is gradually emerging from these studies of the specific molecular events that mediate tubule formation downstream from the initiating inductive process. Since the epithelial conversion of metanephric mesenchyme is believed to be targeted in nephroblastic tumorigenesis, causing the accumulation of blastemal stem cells, these studies should provide clues as to the molecular lesions which allow that to occur. - Cell signaling, Kidney, Differentiation, Growth factors, Induction, Organogenesis, Wilms' tumors, Childhood tumors,
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