Project description:Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, the progenitors in mice terminally differentiate shortly after birth. We defined culture conditions to selectively propagated nephron progenitors in vitro in an undifferentiated state. To understand how expression profiles of Six2+ cells changed during culture in vitro compared with in vivo, we performed microarray analysis of Six2+ cells at E11.5 (starting material) and P0 (experiencing 8 days in vivo), and cultured Six2+ cells at E11.5 for 8 days or 19 days. Microarray analysis were performed with isolated Six2-positive nephron progenitors from transgenic mice embryo at E11.5 or P0, and cultured E11.5 Six2+ cells for 8 or 19 days in conditioned media. P0 non-progenitors represent Six2-GFP-negative cells at P0.
Project description:Self-renewing undifferentiated nephron progenitors express Six2, a transcription factor that is required for their maintenance as undifferentiated progenitors. Differentiation of nephron progenitors is triggered by Wnt/b-catenin signaling. In order to understand how Six2 and Wnt signaling counteract each other, we performed ChIP-seq of Six2 and b-catenin in mesenchymal nephron progenitor cells. Nephron progenitors were FACS-isolated from BAC transgenic Six2GFPcre-positive embryonic kidneys at E16.5. For Six2 ChIP, freshly FACS isolated Six2+ cells were used. For b-catenin ChIP, FACS isolated Six2+ cells were aggregated by centrifugation at 850g for 5min and incubated in 10%FBS/DMEM containing 4uM BIO for 24hrs.
Project description:We have employed whole genome microarray expression profiling to identify genes regulated by Sall1 in the kidney. Six2GFP-positive nephron progenitors and Six2GFP-negative cells were collected from the embryonic kidneys at E15.5 (2 set) , and their expression profiles were compared.
Project description:Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, the progenitors in mice terminally differentiate shortly after birth. We defined culture conditions to selectively propagated nephron progenitors in vitro in an undifferentiated state. To understand how expression profiles of Six2+ cells changed during culture in vitro compared with in vivo, we performed microarray analysis of Six2+ cells at E11.5 (starting material) and P0 (experiencing 8 days in vivo), and cultured Six2+ cells at E11.5 for 8 days or 19 days.
Project description:mIRNA expression profiling of mouse embryonic nephron progenitors at embryonic day 14 isolated by GFP expression driven by Six2-TGC (transgenic mouse line), compared to whole embryonic kidney at day 14
Project description:Eya1 is a critical regulator for establishing nephron fate and acts upstream of Six2. However, how Eya1-centered network operates remains elusive. Here we identified Eya1's interacting factors via mass-spectrometry and show that Eya1 and Six2 interact with Brg1-based SWI/SNF chromatin-remodeling complex in the kidney. Brg1 specifies the nephron fate and regulates Eya1 expression through binding to multiple cis-regulatory-elements. In the nephron-progenitors, Brg1/Six2 co-occupy cis-regulatory-elements at key loci and Brg1-recruitment to many of these regions is disrupted in the absence of Six2. We demonstrate Six2-dependent Brg1-occupancy to Mycn promoter and two distal enhancers of Pbx1, all of which direct nephron-progenitor-specific expression in response to binding to Six2. RNA-seq analysis reveals that depletion of Brg1 leads to upregulation of genes for podocyte-lineages and aberrant activation of cell-death inducing factors. Together, this study highlights an essential function of the Brg1-BAFs in cooperation with Eya1/Six2 in enhancer-mediated regulation of gene expression in the nephron-progenitors.
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: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:SIX2 is expressed by the self-renewing nephron progenitors in the human fetal kidney. We have also discovered that SIX1 is expressed in nephron progenitor population of the human fetal kidney, which is in contrast to the mouse. We performed ChIP-seq of SIX1 and SIX2 in order to identify the target genes of each factor and compare the role that each factor plays in transcriptional regulation of the nephron progenitors. We additionally performed ChIP-seq for p300 and H3K27ac in order to identify active loci and complement the transcription factor data.