Project description:Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. In contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared Six2’s regulatory actions in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Further, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 progenitor activity in the 16 week human fetal kidney. Human SIX1 ChIP-seq revealed a similar set of targets to SIX2, and predicted both factors bind DNA through an identical recognition site. In contrast to the mouse where Six2 binds its own enhancers but doesn’t interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. In contrast, the mouse establishes only an auto-regulatory Six2 loop. It is tempting to speculate that differential SIX-factor regulation may contribute to species differences in the duration of progenitor programs and nephron output.
Project description:p53 limits the self-renewing ability of a variety of stem cells. Here, contrary to its classical role in restraining cell proliferation, we demonstrate a divergent function of p53 in maintenance of self-renewal of the nephron progenitor population in the embryonic mouse kidney. p53-null nephron progenitor cells (NPC) exhibit progressive loss of the self-renewing progenitor niche in the cap mesenchyme, identified by Cited1 and Six2 expression, and loss of cap integrity. Nephron endowment is regulated by NPC availability and their differentiation to nephrons. Quantitatively, the Six2p53-/- cap has 30% fewer Six2GFP+ cells. While the apoptotic index is unchanged the proliferation index is significantly lower, in accordance with cell cycle analysis data showing less mutant Six2p53-/-;GFP+ cells in S and G2/M phases in comparison to Six2p53+/+;GFP+ cells. The mutant kidneys also show nephron deficit and decreased Fgf8 expression. To investigate the underlying changes in gene expression in the cap mesenchyme that contribute to the Six2p53-/- phenotype, we utilized RNA-Seq for transcriptome comparison. Top biological processes affected by p53 loss are development and morphogenesis, cell adhesion/migration, cell survival and metabolism. Cells from the mutant CM showed increased cellular ROS levels as well as deregulated expression of energy metabolism and mitochondrial genes suggesting metabolic dysfunction. Adhesion defects are visualized by decreased immunostaining of adhesion marker NCAM, and may possibly contribute to the differentiation defect as well. Altogether our data suggest a novel role for p53 in enabling self-renewal of the NPC and preservation of the progenitor niche, and thus regulating nephron endowment. mRNA profiles of wild-type (WT) and conditional p53 knockout (KO) of Six2+ mouse nephron progenitor cells (NPC) at embryonic day 15.5
Project description:The regulation of final nephron number in the kidney is poorly understood. However, cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we demonstrate here a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This is accompanied by a clear shift in nephron progenitor identity with a distinct subset of progenitor genes, including Cited1 and Meox1, downregulated, while others were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EDU labelling, an increase in MYC protein and transcriptional upregulation of MYC target genes. Reducing proliferation by introducing Six2 heterozygosity onto the Fgf20-/- background resulted in premature differentiation of the progenitor population. Overall, this data demonstrates a unique dose response of the nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable.
Project description:To characterize the expression profile of mouse nephron progenitors, we performed intracellular labeling of Six2 by antibody, followed by FACS and RNA-seq. To characterize the expression profile of mouse stromal progenitor cells, we isolated Tdt+ cells from Foxd1Cre;Tdt transgenic mice kidney by FACS followed by RNA-seq.
Project description:In developing mammalian kidney, nephron progenitor cells (NPC) give rise to all cells in mature nephrons. Expression of the transcription factor Six2 marks NPC in developing mouse kidneys. Within the Six2+ cell population, uncommitted NPC is marked by Cited1 expression. Towards the depletion of NPC in P2, most of the Cited1+ NPC is committed. Therefore, most of the Six2+ cells in kidney represents committed NPC. In order to explore the mechanism of NPC self-renewal and differentiation, hereby we generated transcriptionl profiles of uncommitted NPC (Cited1RFP+ cells from kidneys of E16.5 Cited1tagRFP transgenic mice) and committed NPC (Six2GFP+ cells from kidneys of P2 Six2TGC transgenic mice).
Project description:To delineate the epigenomic profile of the Six2+ mouse nephron progenitor cells, we mapped open chromatin using ATAC-Seq in Six2+ cells from E16.5 mouse kidneys.
Project description:Kidney development depends on a balance of nephron progenitor self-renewal and commitment. Here we examine how cells shift from a motile progenitor state to a static committed state during nephrogenesis. Cells that express Wnt4, an early marker of commitment, give rise to both the nephron lineage and a migrating sub-population that may re-enter the progenitor pool. Single cell transcriptional profiling reveals Wnt4-lineage cells across a range of transcriptional states, including cells that return to an uninduced progenitor state.