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Polycystic kidney disease (PKD) is a ciliopathy characterized by fluid-filled epithelial cysts in the kidney. Although it is well established that the primary cilium is essential for hedgehog (HH) signaling and HH signaling is abnormally activated in multiple PKD models, the mechanism and function of HH activation in PKD pathogenesis remain incompletely understood. Here we used a transgenic HH reporter mouse line to identify the target tissue of HH signaling in Arl13f/f;Ksp-Cre mutant kidney, in which the cilia biogenesis gene Arl13b is specifically deleted in epithelial cells of the distal nephron. In addition, we used a co-culture system to dissect cross-talk between epithelial and mesenchymal cells in the absence of expanding cysts. Finally, we treated Arl13bf/f;Ksp-Cre mice with the GLI inhibitor GANT61 and analyzed its impact on PKD progression in this model. We found that deletion of Arl13b in epithelial cells in the mouse kidney, in vivo, led to non-cell-autonomous activation of the HH pathway in the interstitium. In vitro, when co-cultured with mesenchymal cells, Arl13b−/− epithelial cells produced more sonic hedgehog in comparison to cells expressing Arl13b. Reciprocally, HH signaling was activated in mesenchymal cells co-cultured with Arl13b−/− epithelial cells. Finally, whole body inhibition of the HH pathway by GANT61 reduced the number of proliferating cells, inhibited cyst progression and fibrosis and preserved kidney function in Arl13bf/f;Ksp-Cre mice. Our results reveal non-cell-autonomous activation of HH signaling in the interstitium of the Arl13bf/f;Ksp-Cre kidney and suggest that abnormal activation of the HH pathway contributes to disease progression.

Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.

The cell surface organelle called the cilium is essential for preventing kidney cyst formation and for establishing left-right asymmetry of the vertebrate body plan. Recent advances suggest that the cilium functions as a sensory organelle in vertebrate cells for multiple signaling pathways such as the hedgehog and the Wnt pathways. Prompted by kidney cyst formation in tuberous sclerosis complex (TSC) patients and rodent models, we investigated the role of the cilium in the TSC-target of rapamycin (TOR) pathway using zebrafish. TSC1 and TSC2 genes are causal for TSC, and their protein products form a complex in the TOR pathway that integrates environmental signals to regulate cell growth, proliferation and survival. Two TSC1 homologs were identified in zebrafish, which we refer to as tsc1a and tsc1b. Morpholino knockdown of tsc1a led to a ciliary phenotype including kidney cyst formation and left-right asymmetry defects. Tsc1a was observed to localize to the Golgi, but morpholinos against it, nonetheless, acted synthetically with ciliary genes in producing kidney cysts. Consistent with a role of the cilium in the same pathway as Tsc genes, the TOR pathway is aberrantly activated in ciliary mutants, resembling the effect of tsc1a knockdown. Moreover, kidney cyst formation in ciliary mutants was blocked by the Tor inhibitor, rapamycin. Surprisingly, we observed elongation of cilia in tsc1a knockdown animals. Together, these data suggest a signaling network between the cilium and the TOR pathway in that ciliary signals can feed into the TOR pathway and that Tsc1a regulates the length of the cilium itself.