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Browsing by Author "Rowe, Steven M."
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Item Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease(Plos, 2016-07-29) Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R.; Leigh, Margaret W.; Zariwala, Maimoona A.; Challa, Anil K.; Keterson, Robert A.; Drummond, Iaian A.; Parant, John M.; Rowe, Steven M.; Hildebrandt, Friedhelm; Porter, Mary E.; Yoder, Bradley K.; Berbari, Nicolas F.; Department of Biology, School of ScienceCiliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.Item Transgenic ferret models define pulmonary ionocyte diversity and function(Springer Nature, 2023) Yuan, Feng; Gasser, Grace N.; Lemire, Evan; Montoro, Daniel T.; Jagadeesh, Karthik; Zhang, Yan; Duan, Yifan; Levlev, Vitaly; Wells, Kristen L.; Rotti, Pavana G.; Shahin, Weam; Winter, Michael; Rosen, Bradley H.; Evans, Idil; Cai, Qian; Yu, Miao; Walsh, Susan A.; Acevedo, Michael R.; Pandya, Darpan N.; Akurathi, Vamsidhar; Dick, David W.; Wadas, Thaddeus J.; Joo, Nam Soo; Wine, Jeffrey J.; Birket, Susan; Fernandez, Courtney M.; Leung, Hui Min; Tearney, Guillermo J.; Verkman, Alan S.; Haggie, Peter M.; Scott, Kathleen; Bartels, Douglas; Meyerholz, David K.; Rowe, Steven M.; Liu, Xiaoming; Yan, Ziying; Haber, Adam L.; Sun, Xingshen; Engelhardt, John F.; Medicine, School of MedicineSpeciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.