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Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

ABSTRACT: BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe conditionally deleted Frs2α in peri-Wolffian duct ST with a Tbx18cre mouse line (Frs2α^ST-/-). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions by three-dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2α^ST-/- embryos had many displaced ureteric bud induction sites when compared with controls. E11.0 Frs2α^ST-/- embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2α^ST-/- mice had higher VUR rates and grades vs.

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Mutant refluxing ureters that inserted improperly into the bladder had shortened intravesicular tunnels (IVTs) when compared with controlsConclusionFrs2α^ST-/- embryos have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths, and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

SUBMITTER: Narla D

PROVIDER: S-EPMC5701656 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

Narla Deepti D Slagle Stacey B SB Schaefer Caitlin M CM Bushnell Daniel S DS Puri Pawan P Bates Carlton M CM

Pediatric research 20170809 6

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe cond ...[more]

PMID: 29135976

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Project description:PurposePax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity.MethodsWe conditionally deleted Fgfr2 in ST (Fgfr2(ST-/-)) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects.ResultsWe confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2(ST-/-) mice at embryonic day (E) 10.5. E11.5 Fgfr2(ST-/-) mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2(ST-/-) mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2(ST-/-) mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes.ConclusionMutations in FGFR2 could possibly cause VUR in humans.

Project description:Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression.

Dataset Information

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

Controls

Publications

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

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