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Project description:Mammalian nephrons originate from a population of nephron progenitor cells, and changes in these cells' transcriptomes contribute to the cessation of nephrogenesis, an important determinant of nephron number. To characterize microRNA (miRNA) expression and identify putative cis-regulatory regions, we collected nephron progenitor cells from mouse kidneys at embryonic day 14.5 and postnatal day zero and assayed small RNA expression and transposase-accessible chromatin. We detect expression of 1104 miRNA (114 with expression changes), and 46,374 chromatin accessible regions (2103 with changes in accessibility). Genome-wide, our data highlight processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways. Furthermore, they identify new candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in nephron progenitor cell differentiation. Finally, we associate expression-changing miRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements and target genes. These analyses highlight new putative cis-regulatory loci for miRNA in nephron progenitors.
| S-EPMC8792369 | biostudies-literature
Project description:Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). smRNA-seq libraries were generated using the QIAseq miRNA library preparation kit (Qiagen 331502), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.
2023-03-04 | GSE168342 | GEO
Project description:Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). ATAC-seq libraries were generated using the Nextera DNA Flex Library Prep Kit (Illumina FC-121-1030) with modifications according to a previously published method (Buenrostro et al., 2013), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.
2023-03-04 | GSE168339 | GEO
Project description:Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development
| PRJNA707113 | ENA
Project description:Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [ATAC-Seq]
| PRJNA707117 | ENA
Project description:Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [miRNA-Seq]
| PRJNA707118 | ENA
Project description:The kidney contains the functional units, the nephrons, surrounded by the renal interstitium. Previously we discovered that, once Six2-expressing nephron progenitor cells and Foxd1-expressing renal interstitial progenitor cells form at the onset of kidney development, descendant cells from these populations contribute exclusively to the main body of nephrons and renal interstitial tissues, respectively, indicating a lineage boundary between the nephron and renal interstitial compartments. Currently it is unclear how lineages are regulated during kidney organogenesis. We demonstrate that nephron progenitor cells lacking Pax2 fail to differentiate into nephron cells but can switch fates into renal interstitium-like cell types. These data suggest that Pax2 function maintains nephron progenitor cells by repressing a renal interstitial cell program. Thus, the lineage boundary between the nephron and renal interstitial compartments is maintained by the Pax2 activity in nephron progenitor cells during kidney organogenesis.
| S-EPMC5532731 | biostudies-literature
Project description:DLG1 (discs-large homolog 1) and CASK (calcium/calmodulin-dependent serine protein kinase) interact at membrane-cytoskeleton interfaces and function as scaffolding proteins that link signaling molecules, receptors, and other scaffolding proteins at intercellular and synaptic junctions. Dlg1-null mice exhibit hydronephrosis, hydroureter, and occasionally hypoplastic kidneys, whereas Cask-null mice do not. To investigate whether DLG1 and CASK cooperate in the developing urogenital system, we generated mice deficient in both DLG1 and CASK either 1) globally, 2) in metanephric mesenchyme, or 3) in nephron progenitors. With each approach, Dlg1;Cask double-knockout (DKO) kidneys were severely hypoplastic and dysplastic and demonstrated rapid, premature depletion of nephron progenitors/stem cells. Several cellular and molecular defects were observed in the DKO kidneys, including reduced proliferation and increased apoptosis of cells in the nephrogenic zone and a progressive decrease in the number of cells expressing SIX2, a transcription factor essential for maintaining nephron progenitors. Fgf8 expression was reduced in early-stage DKO metanephric mesenchyme, accompanied by reduced levels of components of the Ras pathway, which is activated by fibroblast growth factor (FGF) signaling. Moreover, Dlg1(+/-);Cask(-/-) (het/null) kidneys were moderately hypoplastic and demonstrated impaired aggregation of SIX2-positive cells around the ureteric bud tips. Nephron progenitor-specific het/null mice survived with small kidneys but developed glomerulocystic kidney disease and renal failure. Taken together, these results suggest that DLG1 and CASK play critical cooperative roles in maintaining the nephron progenitor population, potentially via a mechanism involving effects on FGF signaling.
| S-EPMC3699830 | biostudies-literature
Project description:The Wilms' tumor suppressor 1 (WT1) gene encodes a DNA- and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, we performed chromatin immunoprecipitation (ChIP) coupled to mouse promoter microarray (ChIP-chip) using chromatin prepared from embryonic mouse kidney tissue. We identified 1663 genes bound by WT1, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, we used a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1(-/-) embryos. Collectively, our results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.
| S-EPMC2835332 | biostudies-literature
Project description:Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.
| S-EPMC10245960 | biostudies-literature