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

AU-rich elements (AREs

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.

No abstract

The Wilms' tumor suppressor gene wt1 encodes a zinc-finger transcription factor that plays an important role in the development of the mammalian genitourinary system. Mutations in WT1 in humans lead to anomalies of kidney and gonad development and cause Wilms' tumor, a pediatric kidney cancer. The inactivation of both wt1 alleles in mice gives rise to multiple organ defects, among them agenesis of kidney, spleen, and gonads. In zebrafish, an ortholog of wt1 has been described that is expressed in the pronephric field and is later restricted to the podocytes. Here, we report the existence of a second wt1 gene in zebrafish, which we have named wt1b (we named the initial gene wt1a). The overall sequence identity of the two Wt1 proteins is 70% and 92% between the zinc-finger regions, respectively. In contrast to wt1a, wt1b is expressed from the earliest stages of development onward, albeit at low levels. Both wt1a and wt1b are expressed in the intermediate mesoderm, with wt1b being restricted to a smaller area lying at the caudal end of the wt1a expression domain. In adult fish, high expression levels for both genes can be found in gonads, kidney, heart, spleen, and muscle. Developmental Dynamics 235:554 -561, 2006.