Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney

The mammalian metanephric kidney is derived from the intermediate mesoderm. In this report, we use molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1⁺ population of metanephric progenitor cells. These include the ureteric epithelium of the collecting duct network, the cap mesenchyme and its nephron epithelia derivatives, the interstitial mesenchyme, vasculature and smooth muscle. Temporal fate mapping shows a progressive restriction of Osr1⁺ cell fates such that at the onset of active nephrogenesis, Osr1 activity is restricted to the Six2⁺ cap mesenchyme nephron progenitors. However, low-level labeling of Osr1⁺ cells suggests that the specification of interstitial mesenchyme and cap mesenchyme progenitors occurs within the Osr1⁺ population prior to the onset of metanephric development. Furthermore, although Osr1⁺ progenitors give rise to much of the kidney, Osr1 function is only essential for the development of the nephron progenitor compartment. These studies provide new insights into the cellular origins of metanephric kidney structures and lend support to a model where Osr1 function is limited to establishing the nephron progenitor pool.     

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.     

Control of kidney development by calcium ions

From the formation of a simple kidney in amphibian larvae, the pronephros, to the formation of the more complex mammalian kidney, the metanephros, calcium is present through numerous steps of tubulogenesis and nephron induction. Several calcium-binding proteins such as regucalcin/SMP-30 and calbindin-D28k are commonly used to label pronephric tubules and metanephric ureteral epithelium, respectively. However, the involvement of calcium and calcium signalling at various stages of renal organogenesis was not clearly delineated. In recent years, several studies have pinpointed an unsuspected role of calcium in determination of the pronephric territory and for conversion of metanephric mesenchyme into nephrons. Influx of calcium and calcium transients have been recorded in the pool of renal progenitors to allow tubule formation, highlighting the occurrence of calcium-dependent signalling events during early kidney development. Characterization of nuclear calcium signalling is emerging. Implication of the non-canonical calcium/NFAT Wnt signalling pathway as an essential mechanism to promote nephrogenesis has recently been demonstrated. This review examines the current knowledge of the impact of calcium ions during embryonic development of the kidney. It focuses on Ca²⁺ binding proteins and Ca²⁺ sensors that are involved in renal organogenesis and briefly examines the link between calcium-dependent signals and polycystins.     

Down-regulated Six2 by knockdown of neurofibromin results in apoptosis of metanephric mesenchyme cells in vitro

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman’s capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.     

Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development

To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, we have used a new mouse line, T-Cre, to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. Our data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis.     

Cessation of renal morphogenesis in mice

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.     

Mdm2 is required for maintenance of the nephrogenic niche

The balance between nephron progenitor cell (NPC) renewal, survival and differentiation ultimately determines nephron endowment and thus susceptibile to chronic kidney disease and hypertension. Embryos lacking the p53-E3 ubiquitin ligase, Murine double minute 2 (Mdm2), die secondary to p53-mediated apoptosis and growth arrest, demonstrating the absolute requirement of Mdm2 in embryogenesis. Although Mdm2 is required in the maintenance of hematopoietic stem cells, its role in renewal and differentiation of stem/progenitor cells during kidney organogenesis is not well defined. Here we examine the role of the Mdm2-p53 pathway in NPC renewal and fate in mice. The Six2-GFP::Cre^tg/+ mediated inactivation of Mdm2 in the NPC (NPC^Mdm2^−/−) results in perinatal lethality. NPC^Mdm2^−/− neonates have hypo-dysplastic kidneys, patchy depletion of the nephrogenic zone and pockets of superficially placed, ectopic, well-differentiated proximal tubules. NPC^Mdm2−/− metanephroi exhibit thinning of the progenitor GFP⁺/Six2⁺ population and a marked reduction or loss of progenitor markers Amphiphysin, Cited1, Sall1 and Pax2. This is accompanied by aberrant accumulation of phospho-γH2AX and p53, and elevated apoptosis together with reduced cell proliferation. E13.5–E15.5 NPC^Mdm2−/− kidneys show reduced expression of Eya1, Pax2 and Bmp7 while the few surviving nephron precursors maintain expression of Wnt4, Lhx1, Pax2, and Pax8. Lineage fate analysis and section immunofluorescence revealed that NPC^Mdm2−/− kidneys have severely reduced renal parenchyma embedded in an expanded stroma. Six2-GFP::Cre^tg/+; Mdm2^f/f mice bred into a p53 null background ensures survival of the GFP-positive, self-renewing progenitor mesenchyme and therefore restores normal renal development and postnatal survival of mice. In conclusion, the Mdm2-p53 pathway is essential to the maintenance of the nephron progenitor niche.     

Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction.

At least fourteen epithelial cell types of the mammalian nephron develop from the metanephric mesenchyme. To distinguish whether this single embryological primordium contains a heterogenous population of committed renal cell lines or a multipotent stem cell, the lac-Z gene was introduced into individual renal progenitors by retroviral mediated gene transfer. The differentiated fate of lac-Z-tagged daughters derived from single metanephric mesenchymal cells was characterized after cytodifferentiation. We found that the metanephric mesenchyme contains multipotent stem cells that can generate at least three distinct cell types; glomerular, proximal and distal epithelia. After induction the fate of this multipotent cell becomes restricted to populate a single nephron segment.     

Kif3a Controls Murine Nephron Number Via GLI3 Repressor,Cell Survival,and Gene Expression in a Lineage-Specific Manner

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3^Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms.     

An epithelial precursor is regulated by the ureteric bud and by the renal stroma

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.     

Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney

During kidney development and in response to inductive signals, the metanephric mesenchyme aggregates, becomes polarized, and generates much of the epithelia of the nephron. As such, the metanephric mesenchyme is a renal progenitor cell population that must be replenished as epithelial derivatives are continuously generated. The molecular mechanisms that maintain the undifferentiated state of the metanephric mesenchymal precursor cells have not yet been identified. In this paper, we report that functional inactivation of the homeobox gene Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia and depletion of the progenitor cell population within the metanephric mesenchyme. Failure to renew the mesenchymal cells results in severe renal hypoplasia. Gain of Six2 function in cortical metanephric mesenchymal cells was sufficient to prevent their epithelial differentiation in an organ culture assay. We propose that in the developing kidney, Six2 activity is required for maintaining the mesenchymal progenitor population in an undifferentiated state by opposing the inductive signals emanating from the ureteric bud.     

Kidney development in cadherin-6 mutants: delayed mesenchyme-to-epithelial conversion and loss of nephrons

During nephrogenesis, dynamic changes in the expression of cell adhesion molecules are evident as epithelial structures differentiate from the induced mesenchyme. The cadherins are thought to play an important role in the metanephric mesenchyme, when cells aggregate to form the renal vesicle, a polarized epithelial structure which eventually fuses with the ureteric bud to generate a continuous nascent nephron. We have generated and analyzed mice with a targeted mutation in the gene encoding cadherin-6 (Cad-6), a type II cadherin expressed during early stages of nephrogenesis. These mice are viable and fertile, and they complete both early and late aspects of nephrogenesis. However, upon closer examination in vitro and in vivo, a fraction of the induced metanephric mesenchyme in Cad-6 mutant kidneys fails to form a fully polarized epithelium on schedule. Moreover, a significant number of the renal vesicles in Cad-6 mutant kidneys apparently fail to fuse to the ureteric bud. These alterations in epithelialization and fusion apparently lead to a loss of nephrons in the adult. These studies support the idea that cadherins play an essential role in the formation of epithelial structures and underscore the importance of timing in orchestrating the morphogenesis of complex epithelial tissues.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.