ABSTRACT: Triplicate pairwise comparsion of FACS sorted GFP+ve Vs GFP-ve cells from the kidneys of the HoxB7-GFP transgenic mice on compugen 22K mouse arrays. HoxB7-GFP mice express GFP in the ureteric tree and its derivatives while the metanephric mesenchyme, interstitium, developing vasculature etc do not. Keywords: repeat sample
Project description:Triplicate pairwise comparsion of FACS sorted GFP+ve Vs GFP-ve cells from the kidneys of the HoxB7-GFP transgenic mice on compugen 22K mouse arrays. HoxB7-GFP mice express GFP in the ureteric tree and its derivatives while the metanephric mesenchyme, interstitium, developing vasculature etc do not.
Project description:Understanding the early developmental processes of the kidney ureteric bud lienage is important for the regeneration of kidney in vitro. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of global gene expression profiles of the developing kidney precursors. Those gene expression data provides insights into not only the stage specific marker genes but also the signals working in each population, which should be informative for the directed differentiation of pluripotent stem cells in vitro. Overall design: Hoxb7-GFP transgenic mice were used to isolate kidney ureteric bud (UB) precursor cells from embryos at E8.75, E9.5, E10.5 and E11.5. For isolation of the pure UB precursor population, dissociated embryos were sorted by the flow cytometry gating Hoxb7-GFP-positive Flk1-negative population. To identify the differences between UB progenitors and neighboring nephron progenitor lineage precursors, the UB precursors were compared with E9.5 Wt1-GFP positive intermediate mesoderm cells, which give rise to the mesonephric and metanephric nephron progenitors.
Project description:The renal vasculature is integral to the physiologic function of the kidneys in regulating hemodynamics of the body and maintaining organ health. The close inter-relationship of capillaries and the renal epithelium is key to renal physiology, but how renal tubules regulate capillary development remains unclear. Our previous work showed that Wnt7b is expressed in the ureteric trunk epithelium and activates canonical Wnt signaling in the surrounding medullary interstitium, where the capillaries reside. In this study, we showed by immunofluorescence that the target interstitial cells of Wnt7b/canonical Wnt signaling are mural cells of periureteric bud capillaries in the nascent renal medulla of embryonic mice. Genetic ablation of Wnt7b enhanced the proliferation of Wnt7b target mural cells, an effect that associated with decreased expression of PDGFR? and p57kip2, a cyclin-dependent kinase inhibitor, in these cells. Furthermore, Wnt7b regulated lumen formation of the capillary endothelium in the renal medulla. In the absence of Wnt7b signaling, the periureteric bud medullary capillaries displayed narrower lumens lined with less flattened endothelial cells and a significantly increased presence of luminal endothelial cell-cell junctions, a transient configuration in the forming blood vessels in the controls. Moreover, the absence of Wnt7b led to greatly diminished levels of vascular endothelial (VE)-cadherin at the cell surface in these blood vessels. VE-cadherin is essential for blood vessel lumen formation; thus, Wnt7b may regulate lumen formation through modulation of VE-cadherin localization. Overall, these results indicate a novel role of Wnt7b signaling and the ureteric bud epithelium in renal medullary capillary development.
Project description:Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.
Project description:Fibroblast growth receptors (FGFRs) consist of four signaling family members. Mice with deletions of fgfr1 or fgfr2 are embryonic lethal prior to the onset of kidney development. To determine roles of FGFR1 and FGFR2 in the ureteric bud, we used a conditional targeting approach. First, we generated transgenic mice using the Hoxb7 promoter to drive cre recombinase and green fluorescent protein expression throughout ureteric bud tissue. We crossed Hoxb7creEGFP mice with mice carrying lox-p sites flanking critical regions of fgfr1 and/or fgfr2. Absence of fgfr1 from the ureteric bud (fgfr1(UB-/-)) results in no apparent renal abnormalities. In contrast, fgfr2(UB-/-) mice have very aberrant ureteric bud branching, thin ureteric bud stalks, and fewer ureteric bud tips. Fgfr2(UB-/-) ureteric bud tips also demonstrate inappropriate regions of apoptosis and reduced proliferation. The nephrogenic mesenchymal lineage in fgfr2(UB-/-) mice develops normal-appearing glomeruli and tubules, and only slightly fewer nephrons than controls. In contrast, fgfr2(UB-/-) kidneys have abnormally thickened subcapsular cortical stromal mesenchyme. Ultimately, fgfr2(UB-/-) adult kidneys are small and abnormally shaped or are hydronephrotic. Finally, there are no additional abnormalities in the fgfr1/2(UB-/-) kidneys versus the fgfr2(UB-/-) kidneys. In conclusion, FGFR2, but not FGFR1, appears crucial for ureteric bud branching morphogenesis and stromal mesenchyme patterning.
Project description:Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. HoxB7-GFP transgenic mice were utilized to isolate the ureteric bud cells from E10.5 embryonic kidneys. The ureteric bud cells were isolated from embryos using microdissection, trypsin treatment and FACS. The RNA was isolated from purified ureteric bud cells and the gene expression profiles were determined by microarrays.
Project description:E11.5 metanephric mesenchyme and ureteric bud were dissected from the E11.5 kidney rudiment using fine manual microdissection (ureteric bud only) or both fine manual microdissection and laser capture microdissection (metanephric mesenchyme) to define the gene expression profiles of these structures. Additionally, HoxA11, HoxD11 compound null E11.5 metanephric mesenchyme was obtained through laser capture microdissection allowing analysis of possible Hox targets in kidney development. Targets from multiple biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430_v2 arrays. Using microdissection techniques, ureteric bud and metanephric mesenchyme were dissected from E11.5 kidney rudiments allowing the identificated genes specifically regulated in either structure. In addition, Hoxa11, Hoxd11 compound null E11.5 metanephric mesenchyme were normalized to wild type embryonic controls allowing the identification of potential Hox targets in normal kidney development. Each structure/genotype were represented in biological (seperate embryo) replicate.
Project description:BACKGROUND: Genetic analysis in the mouse revealed that GREMLIN1 (GREM1)-mediated antagonism of BMP4 is essential for ureteric epithelial branching as the disruption of ureteric bud outgrowth and renal agenesis in Grem1-deficient embryos is restored by additional inactivation of one Bmp4 allele. Another BMP ligand, BMP7, was shown to control the proliferative expansion of nephrogenic progenitors and its requirement for nephrogenesis can be genetically substituted by Bmp4. Therefore, we investigated whether BMP7 in turn also participates in inhibiting ureteric bud outgrowth during the initiation of metanephric kidney development. METHODOLOGY/PRINCIPAL FINDINGS: Genetic inactivation of one Bmp7 allele in Grem1-deficient mouse embryos does not alleviate the bilateral renal agenesis, while complete inactivation of Bmp7 restores ureteric bud outgrowth and branching. In mouse embryos lacking both Grem1 and Bmp7, GDNF/WNT11 feedback signaling and the expression of the Etv4 target gene, which regulates formation of the invading ureteric bud tip, are restored. In contrast to the restoration of ureteric bud outgrowth and branching, nephrogenesis remains aberrant as revealed by the premature loss of Six2 expressing nephrogenic progenitor cells. Therefore, very few nephrons develop in kidneys lacking both Grem1 and Bmp7 and the resulting dysplastic phenotype is indistinguishable from the one of Bmp7-deficient mouse embryos. CONCLUSIONS/SIGNIFICANCE: Our study reveals an unexpected inhibitory role of BMP7 during the onset of ureteric bud outgrowth. As BMP4, BMP7 and GREM1 are expressed in distinct mesenchymal and epithelial domains, the localized antagonistic interactions of GREM1 with BMPs could restrict and guide ureteric bud outgrowth and branching. The robustness and likely significant redundancy of the underlying signaling system is evidenced by the fact that global reduction of Bmp4 or inactivation of Bmp7 are both able to restore ureteric bud outgrowth and epithelial branching in Grem1-deficient mouse embryos.