The Fate and Behavior of Ret-expressing Tip Cells in Kidney Development

The Fate and Behavior of Ret-expressing Tip Cells in Kidney Development Paul N. Riccio The mammalian kidney is a complex structure composed of many highly differentiated cell types. The spatial distribution of these cells, however, emerges from elaboration of an earlier program of epithelial and mesenchymal interactions in which a tubular epithelium, the ureteric bud, undergoes successive rounds of branching within the metanephric mesenchyme. Signaling through the receptor tyrosine kinase RET is required for the normal progression of this developmental program. A complete portrait of the transcriptional changes effected by RET activation has emerged through microarray profiling. Genetic studies in the mouse have further elucidated the roles of many of these downstream genes in mediating particular inductive events, or in directing a cell toward a particular differentiation program. Comparatively little is known, however, about the fates of Ret-expressing tip cells, themselves, and of the cell-level manipulations required to sustain branching of the ureteric bud. The research presented in this thesis is an attempt to broach these questions by harnessing the growing precision of cell-level, mosaic genetic manipulations. An inducible Cre allele under control of the Ret promoter served as a tool to unambiguously confirm that Ret-expressing tip cells are multipotent progenitors that give rise to the entirety of the renal collecting system. The Ret-expressing tip cells form a self-renewing niche that furthermore gives rise to the “trunk” regions of collecting system. As these tip-derived progeny differentiate, they are competent to assume both principal and intercalated phenotypes. The RetCreERT2 allele, itself a null allele, was used to mosaically ablate Ret within the tip domain by crossing to a conditional Ret line. Loss of Ret from a portion of the tip cells severely disrupted normal branching, yielding hypomorphic kidneys with dysplastic tips. This occurred even upon later stage Ret deletion, suggesting a continued role for Ret in maintaining the branching program. Surprisingly, the mosaic loss of Ret from the tip domain is more disruptive to the branching program than is mosaic deletion of tip cells themselves. In the latter set of experiments, kidney growth was reduced, however, the morphology of the tips and of the collecting system remained normal. Mosaic analysis with double markers (MADM) was utilized to follow the fates of individual cells that lost Ret activity. This genetic tool proved incredibly powerful, revealing that tip cells that lose Ret are near completely excluded from the tip domain. Initial results suggest that this sorting behavior might be fairly rapid, which would reject the hypothesis that Ret confers a proliferative advantage to the cells. Ret activity might instead confer an ability to undergo cell rearrangements or migration-like movements that keep a cell positioned at the tip. Collectively, these findings augment our appreciation of the Ret-expressing tip domain as a special compartment within the branching ureteric bud. Ret plays a continued role in maintaining branching past its previously known role in primary ureteric bud formation. Finally, the observations made using the MADM technique compel the hypothesis that Retdependent cell rearrangements sculpt and continually refine the branching ureteric bud.