Studies of terminal epithelial differntiation in the kidney, the genesis of cell polarity and the development of glomerular structure, have until now been limited to in vitro cell culture models or to biochemical approaches. Zebrafish pronephric kidney mutants offer a unique opportunity to apply a genetic approach to the discovery of essential genes that are required for differentiated epithelial cell function. Fifteen mutations have been isolated which give rise to cysts in the pronephos, resulting from defects in terminal epithelial differentiation. Glomerular structure and epithelial cell polarity are deranged into two mutants, double bubble and bazooka joe as judged by the apical expression of the Na+-K+ATPase. We will test the hypothesis that cell-cell and cell-matrix interactions are determinants of epithelial polarity in vivo by examining the distribution of cadherins, integrins and matrix molecules in double bubble, bazooka joe, and similar mutants using electron microscopy and immunocytochemical methods. Secondly, we hypothesize that defects in terminal differentiation can arise either when extracellular signals are missing or when the interpretation of those signals by cellular signal transduction mechanisms are defective. Therefore, we will construct, by cell transplantation, chimeric embryos containing labeled wild=type cells in the mutant double bubble background (and vice versa) and then test whether mutations affecting epithelial differentiation act in a cell-autonomous or non-autonomous fashion using the Na+ -K+ ATPase as marker of cell polarity. Finally we will use the existing Ssr map of the zebrafish gone=ome to map the 15 pronephiric mutations and create a physical framework for the future cloning of mutated genes. The formation of cysts in this set of mutants suggests parallels to polycystic kidney disease where loss of differentiated cell function and cell polarity are thought to play a role in cytogenesis. The multilocus nature of PKD suggests that a variety of factors are necessar for the establishment of cell polarity and function. It is likely that the 15 genes whe have defined in zebrafish form the basis of a genetic pathway underlying the terminal differentiation of epithelial cells.
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