Dataset Information

Neuropeptide Y functions as a facilitator of GDNF-induced budding of the Wolffian duct.

ABSTRACT: Ureteric bud (UB) emergence from the Wolffian duct (WD), the initiating step in metanephric kidney morphogenesis, is dependent on GDNF; however, GDNF by itself is generally insufficient to induce robust budding of the isolated WD in culture. Thus, additional factors, presumably peptides or polypeptide growth factors, might be involved. Microarray data from in vivo budding and non-budding conditions were analyzed using non-negative matrix factorization followed by gene ontology filtering and network analysis to identify sets of genes that are highly regulated during budding. These included the GDNF co-receptors GFRalpha1 and RET, as well as neuropeptide Y (NPY). By using ANOVA with pattern matching, NPY was also found to correlate most significantly to the budded condition with a high degree of connectedness to genes with developmental roles. Exogenous NPY [as well as its homolog, peptide YY (PYY)] augmented GDNF-dependent budding in the isolated WD culture; conversely, inhibition of NPY signaling or perturbation of NPY expression inhibited budding, confirming that NPY facilitates this process. NPY was also found to reverse the decreased budding, the downregulation of RET expression, the mislocalization of GFRalpha1, and the inhibition of AKT phosphorylation that resulted from the addition of BMP4 to the isolated WD cultures, suggesting that NPY acts through the budding pathway and is reciprocally regulated by GDNF and BMP4. Thus, the outgrowth of the UB from the WD might result from a combination of the upregulation of the GDNF receptors together with genes that support GDNF signaling in a feed-forward loop and/or counteraction of the inhibitory pathway regulated by BMP4.

SUBMITTER: Choi Y

PROVIDER: S-EPMC2781055 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Neuropeptide Y functions as a facilitator of GDNF-induced budding of the Wolffian duct.

Choi Yohan Y Tee James B JB Gallegos Thomas F TF Shah Mita M MM Oishi Hideto H Sakurai Hiroyuki H Kitamura Shinji S Wu Wei W Bush Kevin T KT Nigam Sanjay K SK

Development (Cambridge, England) 20091201 24

Ureteric bud (UB) emergence from the Wolffian duct (WD), the initiating step in metanephric kidney morphogenesis, is dependent on GDNF; however, GDNF by itself is generally insufficient to induce robust budding of the isolated WD in culture. Thus, additional factors, presumably peptides or polypeptide growth factors, might be involved. Microarray data from in vivo budding and non-budding conditions were analyzed using non-negative matrix factorization followed by gene ontology filtering and netw ...[more]

PMID: 19934016

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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.

Project description:Introduction and objectives: Proper In utero Wolffian duct (WD) development requires androgens and is essential to epididymis formation and male fertility. However, non-hormonal factors that control WD homeostasis and development remain largely unknown. In this study, we investigated the contribution of Hedgehog signaling pathway to Wolffian duct development by combining pharmacological approaches on organotypic cultures of WD with microarray profiling. Methods: WD were collected from embryos isolated from 16.5 days post-coitum pregnant mice and cultured in air-liquid condition at 37oC during 72 hours in the presence of either: 1) Cyclopamine (25 µM): Hh inhibitor; 2) Smoothened agonist (0.5 µM ; SAG): Hh activator; 3) Culture media (control). WD pictures were taken and analyzed on ImageJ. At the end of the culture, WD were snap-frozen to perform transcriptomic microarray analyses. Three to four biological replicates per condition, i.e. control (n=3), SAG (n=4) and cyclopamine (n=3), were used for microarray analyses. The quality of the RNA was determined using an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and displayed a RIN ranging from 9.10 to 9.60 out of 10 for the different samples. Microarray analyses were carried out on Affymetrix Mouse Clariom S arrays (Thermofisher) according to the Affymetrix standard protocol. In brief, 100 ng total RNA samples were labeled using the GeneChip® WT Plus Reagent Kit protocol and hybridized to the arrays as described by the manufacturer (Affymetrix, Thermofisher). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After a 16-h hybridization period, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol. The arrays were scanned using the Affymetrix GCS 3000 7G and Gene-Chip Command Console Software (AGCC) (Affymetrix, Thermofisher) to produce the probe cell intensity data (CEL). The imaged data were then analyzed using the Affymetrix Expression Console Software to perform the quality control, background subtraction and normalization of probe set intensities using Robust Multiarray Analysis (RMA). Microarray processing was performed by the Gene Expression Core facility of the Genomic platform of the Centre Hospitalier Universitaire de Quebec Research Center. The microarray CEL files were imported and analyzed with Transcriptome Analysis Console (TAC) Software 4.0.2 (Applied Biosystems), and submitted to RMA normalization. Samples were included within three different treatment groups (control, SAG and cyclopamine) and compared by Analysis of Variance. Principal Component Analysis and heat maps were performed with Partek Pathway (Partek Incorporated). Biological pathways analyses were performed by using 5 distinct algorithms (i.e. DAVID, STRING, GOrilla, Metascape, GSEA and BLAST2GO). Conclusion: The data made available on GEO repository provide new insights regarding the mechanisms that control epididymis morphogenesis and male fertility. Financially supported by a CIHR grant to CB.

Dataset Information

Neuropeptide Y functions as a facilitator of GDNF-induced budding of the Wolffian duct.

Publications

Neuropeptide Y functions as a facilitator of GDNF-induced budding of the Wolffian duct.

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