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.

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The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney.

Galactose-activated transcription of the Saccharomyces cerevisiae GAL genes occurs when Gal3 binds the Gal4 inhibitor, Gal80. Noninteracting variants of Gal3 or Gal80 render the GAL genes noninducible. To identify the binding determinants for Gal3's interaction with Gal80 we carried out GAL3-GAL80 intergenic suppression analyses and selected for new GAL3 mutations that impair the Gal3-Gal80 interaction. We show that a GAL3 C -D368V mutation can suppress the noninducibility due to a GAL80 S-1 -G323R mutation, and a GAL80-M350C mutation can suppress the noninducibility due to a gal3-D111C mutation. A reverse two-hybrid selection for GAL3 mutations that impair the Gal3-Gal80 interaction yielded 12 singleamino-acid substitutions at residues that are predicted to be surface exposed on Gal3. The majority of the affected Gal3 residues localized to a composite surface that includes D111 and a sequence motif containing D368, which has been implicated in interaction with Gal80. The striking colocalization of intergenic suppressor residues and Gal80 nonbinder residues identifies a Gal3 surface that likely interacts with Gal80.

Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3 C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3 SOC , and showed that it suppressed a new GAL3 C allele. We tested the effect of GAL3 SOC on several newly isolated and existing GAL3 C alleles that map throughout the gene. All except one GAL3 C allele, D368V, were suppressible by GAL3 SOC. GAL3 SOC and all GAL3 C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose-and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.

High nitrate levels in the environment may result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases 1 . However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease 2 . Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells 3 . We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue -the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine. Video LinkThe video component of this article can be found at https://www.jove.com/video/2725/ Protocol 1. Conditioning of non-transgenic recipient fish.