Role of Prickle1 in Ciliogenesis and Localization of Sonic Hedgehog Components

Robinow Syndrome (RS) is a rare genetic disorder characterized by dwarfism, craniofacial dysmorphology, vertebral and gonadal dystrophy. RS is caused by mutations in members of the Wnt/Planar Cell Polarity pathway. In addition, patients with RS have phenotypic features of ciliopathies. To model RS, we have chosen Prickle1Bj/Bj mice. The Prickle1Bj/Bj develop wider faces and shorter limbs similar to many mouse models of RS and ciliopathies. During facial development, I observed swollen primary cilia and increased Hedgehog (HH) signaling. Importantly, I localized Prickle1 protein in the nucleus and cilia of cells in the Prickle1+/+ and Prickle1Bj/Bj embryonic faces. The primary cilium is a microtubule-based organelle that perceives and executes mechanical and chemical signaling transduction for the HH pathway. Mediating the normal activity of HH requires an intact ciliary axoneme and appropriate localization of HH components by intraflagellar transport (IFT) in the cilium. In Prickle1Bj/Bj mutants, I observed widening of the ciliary pockets with Transmission Electron Microscopy. Immunofluorescence staining revealed defective anterograde (IFT-88, IFT-52) and retrograde (IFT-122, IFT-140) intraflagellar trafficking in addition to the mislocalization of a ciliary membrane component (ARL13B) in the Prickle1Bj/Bj face compared to controls. I observed increased staining of SMO and Gli2, in the Prickle1Bj/Bj nucleus and ciliary axoneme. In agreement with these results, I observed defective Gli processing by Western Blot in the Prickle1Bj/Bj face and limbs. We attempted to rescue the Prickle1Bj/Bj phenotype by dampening HH signaling with Vismodegib, an FDA-approved HH antagonist, and observed tissue specific responses in the skeleton, and ciliary morphology. We directly tested if HH signaling is contributing to the etiology of RS using primary RS fibroblast cells. In RS fibroblasts Prickle1 and SMO proteins are localized to the ciliary axoneme. We modulated HH signaling in the RS-fibroblasts using Vismodegib, and purmorphamine (agonist) which resulted in shortened cilia enriched in SMO. I observed reduced Gli processing in response to SHH treatment in the RS fibroblasts. In conclusion, my data suggests that the Prickle1Bj/Bj facial phenotype arises from increased HH signaling resulting from defective intraflagellar transport, and defective Gli processing and this mechanism contributes to the etiology of RS