Hedgehog gene regulatory networks in the mammalian kidney

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Project Summary/Abstract Mesenchymal nephron progenitor cells (MNPs) give rise to all nephron tubules in the mammalian kidney. Premature depletion of these cells leads to low nephron numbers, increasing the risk of high blood pressure and various renal diseases. In order to intervene in premature depletion of MNPs, we must better understand how the MNP population is maintained during development. To date, the processes required for the maintenance of MNPs in vivo are poorly understood. Our preliminary data suggest that Hedgehog signaling plays important roles in MNP maintenance. Hedgehog signaling regulates numerous developmental processes. Upon binding of Hedgehog to Patched, Smoothened (Smo) is relieved from Patched-mediated inhibition, initiating the signaling cascade. To test the potential role of Hedgehog signaling in MNPs, we generated Smo mutant kidneys with Six2Cre and found that MNPs lacking Smo were prematurely depleted during development, resulting in a 50% reduction in nephron number. Our transcriptional profiling data from Smo loss- and gain-of-function mutant MNPs show a linear correlation between Ptch1 expression and Smo dosage, suggesting that MNPs do respond to Hedgehog signaling. Our data show that Fox transcription factors are downstream targets of Hedgehog signaling in MNPs and that loss of Hedgehog signaling results in the activation of Notch signaling, a major differentiation signal for MNPs. Based on our findings, we hypothesize that ligand-dependent Hedgehog signaling maintains MNPs by repressing Notch signal and activating Fox genes. We propose to (1) perform genetic analyses to determine if Hedgehog signaling increases self-renewal of MNPs and nephron endowment, (2) test if genetic attenuation or pharmacological inhibition of Notch signaling rescues premature depletion of Smo mutant MNPs, and (3) test if loss of Fox transcription factors results in premature depletion of MNPs and lower nephron numbers. Successful completion of these aims will (1) provide novel insights into how Hedgehog signaling impacts nephron endowment, (2) identify direct target genes operating downstream of Hedgehog signaling in MNPs, (3) determine if Fox transcription factors play key roles in nephrogenesis, and (4) improve in vitro renal organoid cultures.