Project description:Successive activation of Wnt4 and Notch2 generates nephrons from the metanephric mesenchyme. Mesenchymal-to-epithelial transition requires Wnt4, and normal development of the proximal nephron (epithelia of glomeruli and proximal tubules) requires Notch2. It is unknown, however, whether Notch2 dictates the fate of the proximal nephron directly. Here, we generated a mutant strain of mice with activated Notch2 in Six2-containing nephron progenitor cells of the metanephric mesenchyme. Notch2 activation did not skew the cell fate toward the proximal nephron but resulted in severe kidney dysgenesis and depletion of Six2-positive progenitors. We observed ectopic expression of Wnt4 and premature tubule formation, similar to the phenotype of Six2-deficient mice. Activation of Notch2 in the progenitor cells suppressed Pax2, an upstream regulator of Six2, possibly through Hesr genes. Taken together, these data suggest that a positive feedback loop exists between Notch2 and Wnt4, and that Notch2 stabilizes, rather than dictates, nephron fate by shutting down the maintenance of undifferentiated progenitor cells, thereby depleting this population.
Project description:Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.
Project description:Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.
Project description: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(Mdm)2(-/-)) results in perinatal lethality. NPC(Mdm)2(-/-) 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-?H2AX 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.
Project description:BACKGROUND:Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear. METHODS:We used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development. RESULTS:We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells (in contrast to deletion of Dnmt3a or Dnm3b) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. CONCLUSIONS:These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.
Project description:Kidney nephrons are composed of proximal and distal tubule segments that perform unique roles in excretion. The developmental pathways that establish nephron segment identities from renal progenitors are poorly understood. Here, we used the zebrafish pronephros to study nephron segmentation. We found that zebrafish nephron progenitors undergo elaborate spatiotemporal expression changes of many genes before adopting a segment fate. Initially, two domains of nephron progenitors are established, then are subdivided and demarcate individual nephron segments. Using genetic and chemical genetic models of retinoic acid (RA) deficiency, we discovered that RA modulates rostral progenitor formation. To delineate downstream pathways, we knocked down the irx3b transcription factor and found it regulates proximal tubule segment size and distal segment differentiation. Our results suggest a model whereby RA patterns the early field of nephron progenitors, with subsequent factors like irx3b acting to refine later progenitor subdomains and ensure activation of segment-specific gene programs.
Project description: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.
Project description:Cellular binary fate decisions require the progeny to silence genes associated with the alternative fate. The major subsets of alpha:beta T cells have been extensively studied as a model system for fate decisions. While the transcription factor RUNX3 is required for the initiation of Cd4 silencing in CD8 T cell progenitors, it is not required to maintain the silencing of Cd4 and other helper T lineage genes. The other runt domain containing protein, RUNX1, silences Cd4 in an earlier T cell progenitor, but this silencing is reversed whereas the gene silencing after RUNX3 expression is not reverse. Therefore, we hypothesized that RUNX3 and not RUNX1 recruits other factors that maintains the silencing of helper T lineage genes in CD8 T cells. To this end, we performed a proteomics screen of RUNX1 and RUNX3 to determine candidate silencing factors.
Project description:Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.