Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation 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.

Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate.

Neural stem cell (NSC) maintenance and functions are regulated by reactive oxygen species (ROS). However, the mechanisms by which ROS control NSC behavior remain unclear. Here we report that ROS-dependent Igfbp2 signaling controls DNA repair pathways which balance NSC self-renewal and lineage commitment. Ncf1 or Igfbp2 deficiency constrains NSCs to a self-renewing state and prevents neurosphere formation. Ncf1-dependent oxidation of Igfbp2 promotes neurogenesis by NSCs in vitro and in vivo while repressing Brca1 DNA damage response genes and inducing DNA double-strand breaks (DDSBs). By contrast, Ncf1-/- and Igfbp2-/- NSCs favor the formation of oligodendrocytes in vitro and in vivo. Notably, transient repression of Brca1 DNA repair pathway genes induces DDSBs and is sufficient to rescue the ability of Ncf1-/- and Igfbp2-/- NSCs to lineage-commit to form neurospheres and neurons. NSC lineage commitment is dependent on the oxidizable cysteine-43 residue of Igfbp2. Our study highlights the role of DNA damage/repair in orchestrating NSC fate decisions downstream of redox-regulated Igfbp2.

Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury.

Cartilaginous airways of larger mammals and the mouse trachea contain at least 3 well-established stem cell compartments, including basal cells of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline with age in mice. Notably, Sox9-lineage glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair, and its deletion significantly reduced proliferation following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation of progenitors in both the SAE and SMG shortly following injury, but these progenitors failed to proliferate in vitro in the absence of Sox9, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not surface airway, progenitors. We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively.

Ferret models of alpha-1 antitrypsin deficiency develop lung and liver disease.

Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.

Lef-1 controls cell cycle progression in airway basal cells to regulate proliferation and differentiation.

The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine.

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b.

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.

Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity.

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.

Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus.

The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 "safe harbor" locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG-LoxPtdTomatoStopLoxPEGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.