Loss of kidney function underlies many renal diseases1. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones2,3. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury4,5, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10–30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

The Wilms tumor protein WT1 is an essential factor for kidney development. In humans, mutations in WT1 lead to Wilms tumor, a pediatric kidney cancer as well as to developmental anomalies concerning the urogenital tract. Inactivation of Wt1 in mice causes multiple organ defects most notably agenesis of the kidneys. In zebrafish, two paralogous wt1 genes exist, wt1a and wt1b. The wt1 genes are expressed in a similar and overlapping but not identical pattern. Here, we have examined the role of both wt1 genes in early kidney development employing a transgenic line with pronephros specific GFP expression and morpholino knockdown experiments. Inactivation of wt1a led to failure of glomerular differentiation and morphogenesis resulting in a rapidly expanding general body edema. In contrast, knockdown of wt1b was compatible with early glomerular development. After 48 h, however, wt1b morphant embryos developed cysts in the region of the glomeruli and tubules and subsequent pericardial edema at 4 days post-fertilization. Thus, our data suggest different functions for wt1a and wt1b in zebrafish nephrogenesis. While wt1a has a more fundamental and early role in pronephros development and is essential for the formation of glomerular structures, wt1b functions at later stages of nephrogenesis.

The zebrafish is a valuable vertebrate model for kidney research. The majority of previous studies focused on the zebrafish pronephros, which comprises only two nephrons and is structurally simpler than the mesonephros of adult fish and the metanephros of mammals. To evaluate the zebrafish system for more complex studies of kidney development and regeneration, we investigated the development and postinjury regeneration of the mesonephros in adult zebrafish. Utilizing two transgenic zebrafish lines (wt1b::GFP and pod::NTR-mCherry), we characterized the developmental stages of individual mesonephric nephrons and the temporal-spatial pattern of mesonephrogenesis. We found that mesonephrogenesis continues throughout the life of zebrafish, with a rapid growth phase during the juvenile period and a slower phase in adulthood such that the total nephron number of juvenile and adult fish linearly correlates with body mass. Following gentamicin-induced renal injury, the zebrafish mesonephros can undergo de novo regeneration of mesonephric nephrons, a process known as neonephrogenesis. We found that wt1b expression was induced in individually dispersed cells in the mesonephric interstitium as early as 48 h following injury. These wt1b-expressing cells formed aggregates by 72-96 h following injury which proceeded to form nephrons. This suggests that wt1b may serve as an early marker of fated renal progenitor cells. The synchronous nature of regenerative neonephrogenesis suggests that this process may be useful for studies of nephron development.

Fat ͉ planar cell polarity ͉ polycystic kidney disease

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