Zfp423 regulates Sonic hedgehog signaling via primary cilium function

Zfp423 encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. Here we show that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh, and primary cilia abnormalities that include diminished frequency of both Smoothened and IFT88 localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3. Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia.\n\nAuthor SummaryCiliopathies are a broad group of individually rare genetic disorders that share overlapping phenotypes and mutations in genes that make components of the primary cilium. Mutations in ZNF423 are an exception. Patients and mouse models show characteristic hypoplasia of the cerebellar midline (Joubert syndrome), but the gene encodes a nuclear transcription factor. The mouse gene, Zfp423, is expressed in a dynamic developmental pattern, leaving the cellular mechanism for this brain malformation unresolved. One report suggested reduced Purkinje cell expression of Shh, a key mitogen for cerebellar granule cell precursors (GCPs) whose signal transduction occurs at the primary cilium, as the key event. We show that Zfp423 mutants expressed normal Shh levels, but that Zfp423-depleted GCPs were unable to respond. Primary cilia on Zfp423-mutant GCPs in situ typically had a wider base and longer extension. ZNF423-depletion in a human cell culture model resulted in defective translocation of Smoothened, a key event in Shh signaling, and of the intraflagellar transport protein IFT88. RNA-Seq and RT-qPCR experiments identified known ciliopathy genes as potential conserved targets of ZNF423 and Zfp423. One of these, TULP3, was both up-regulated in ZNF423/Zfp423-deficient cells and directly bound by Zfp423 in granule cell precursors. Reversing the overexpression of TULP3 in ZNF423-depleted human cell culture model reversed the defect in Smoothened translocation.