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Item Loss of OCRL increases ciliary PI(4,5)P2 in Lowe oculocerebrorenal syndrome(The Company of Biologists, 2017-10-15) Prosseda, Philipp P.; Luo, Na; Wang, Biao; Alvarado, Jorge A.; Hu, Yang; Sun, Yang; Ophthalmology, School of MedicineLowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts and glaucoma, mental retardation, and proximal renal tubular dysfunction. Mutations in OCRL, an inositol polyphosphate 5-phosphatase that dephosphorylates PI(4,5)P2, cause Lowe syndrome. Previously we showed that OCRL localizes to the primary cilium, which has a distinct membrane phospholipid composition, but disruption of phosphoinositides in the ciliary membrane is poorly understood. Here, we demonstrate that cilia from Lowe syndrome patient fibroblasts exhibit increased levels of PI(4,5)P2 and decreased levels of PI4P. In particular, subcellular distribution of PI(4,5)P2 build-up was observed at the transition zone. Accumulation of ciliary PI(4,5)P2 was pronounced in mouse embryonic fibroblasts (MEFs) derived from Lowe syndrome mouse model as well as in Ocrl-null MEFs, which was reversed by reintroduction of OCRL. Similarly, expression of wild-type OCRL reversed the elevated PI(4,5)P2 in Lowe patient cells. Accumulation of sonic hedgehog protein in response to hedgehog agonist was decreased in MEFs derived from a Lowe syndrome mouse model. Together, our findings show for the first time an abnormality in ciliary phosphoinositides of both human and mouse cell models of Lowe syndrome.Item Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis(BMC, 2012-11-01) Pasek, Raymond C.; Berbari, Nicolas F.; Lewis, Wesley R.; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of ScienceBackground: Clusterin associated protein 1 (CLUAP1) was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarily conserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice. Methods: Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, β-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos. Results: Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed. Conclusions: These data indicate that the function of Cluap1 is evolutionarily conserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an intraflagellar transport B complex protein. Future studies on mammalian Cluap1 utilizing this mouse model may provide insights into the role for Cluap1 in intraflagellar transport and the association with colon cancer and cystic kidney disorders.Item Sonic Hedgehog Signaling in Inner Ear Organoid Development(2019-08) Longworth-Mills, Emma; Hashino, Eri; Jones, Kathryn; Robling, Alexander; Zimmers, Teresa; Chen, JinhuiLoss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development.