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

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 nonbudding 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 GFRα1 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 GFRα1, 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.