While many factors responsible for tissue patterning and cell fate determination have been defined, we lack a comprehensive understanding of the specific molecules that mediate differentiation in any given tissue, the responses that each factor elicits, the necessary tissue interactions, and the sequence of events required for proper patterning. In pursuit of this goal, we have devised cell and explant culture systems for producing and defining the factors involved in kidney development, are currently purifying those factors, and plan to analyze molecular responses to the factors in organogenesis. Previously, we formulated culture conditions for the maintenance and growth of cells from the ureteric bud, which forms the collecting duct in the adult. These cells can be continuously propagated in serum-free defined medium and have retained their inductive activity for metanephric mesenchyme. The inductive factor(s) can be collected from conditioned medium and has been partially purified. For metanephric mesenchyme, stem cells of the epithelial structures in the nephron, we previously showed that Fgf2 can mediate the early events of differentiation but not epithelial conversion and tubule formation. However, now we report that isolated undifferentiated mesenchymes can be induced with our bud cell line- derived factor(s) and Fgf2 to undergo complete nephrogenesis. This clearly demonstrates that induction in the kidney is mediated by secreted soluble factors and does not require direct cell-cell interaction as previously thought. Since this morphogenesis occurs over a 3-day period, it is possible to stop and evaluate differentiation in cultured mesenchymes at any point. Because there is no contaminating inductive tissue, differential display techniques can be applied to the homogeneous explants. Thus far, this approach has yielded a series of sequences which are expressed in induced tubular cultures but not condensed cultures and vice versa. Some are known cytoskeletal components, while others appear to be novel sequences. Finally, we are evaluating the nature of inductive molecules in renal embryonal tumors, i.e., nephroblastomas, since these tumors caricature developing kidneys. Their histologic profiles suggest that lesions have occurred in signaling pathways which have restricted the ability of the nephrogenic stem cells to completely differentiate. Accordingly, we are evaluating the expression of specific renal signaling factors in tumors and have observed a deficiency in a putative renal inductive factor bmp7.
Showing the most recent 10 out of 11 publications
