Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63⁺ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.     

Activation of NOTCH1 or NOTCH3 Signaling Skews Human Airway Basal Cell Differentiation toward a Secretory Pathway

Airway basal cells (BC) function as stem/progenitor cells capable of differentiating into the luminal ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with γ-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secretory and ciliated cells, but more so for the secretory lineage. Sustained cell autonomous ligand independent Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the NOTCH2 and 4 pathways have little effect on BC differentiation into secretory and ciliated cells, while activation of the NOTCH1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment.     

Stabilized β-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis

The precise mechanisms by which β-catenin controls morphogenesis and cell differentiation remain largely unknown. Using embryonic lung development as a model, we deleted exon 3 of β-catenin via Nkx2.1-cre in the Catnb[+/lox(ex3)] mice and studied its impact on epithelial morphogenesis. Robust selective accumulation of truncated, stabilized β-catenin was found in Nkx2.1-cre;Catnb[+/lox(ex3)] lungs that were associated with the formation of polyp-like structures in the trachea and main-stem bronchi. Characterization of polyps suggests that accumulated β-catenin impacts epithelial morphogenesis in at least two ways. “Intracellular” accumulation of β-catenin blocked differentiation of spatially-appropriate airway epithelial cell types, Clara cells, ciliated cells and basal cells, and activated UCHL1, a marker for pulmonary neuroendocrine cells. There was also evidence for a “paracrine” impact of β-catenin accumulation, potentially mediated via activation of Bmp4 that inhibited Clara and ciliated, but not basal cell differentiation. Thus, excess β-catenin can alter cell fate determination by both direct and paracrine mechanisms.     

Isolation and Characterization of Basal Cells from Human Upper Respiratory Epithelium

Cellular pathways of normal and reparative differentiation of upper airway epithelium are not well understood. Of the three main cell types, basal and secretory cells are known to divide, while ciliated cells are considered terminally differentiated. Several investigations support the role of the basal cell as a progenitor cell type, but others suggest that the secretory cell can regenerate a complete mucocilliary epithelium. Thus, lineage relationships within renewing adult epithelia are still unclear. Understanding the pathways involved in upper airway epithelial cell differentiation is critical for studying injury and repair mechanisms and for developing clinical strategies for tracheal reconstruction. We undertook the current studies to determine the integrin profile of isolated human upper airway basal cells. Respiratory epithelial cells (REC) were isolated by elastase digestion, stained with FITC-labeledGriffonia simplicifoliaisolectin B₄(GSI-B₄), and sorted by flow cytometry. Approximately 80% of the lectin-positive cells were basal cells, as determined by morphology and cytokeratin staining. These cells expressed integrins α₁, α₂, α₃, α₅, α_vβ₅, β₁, β₃, and α₆β₄, by immunohistochemistry. This is the first report to identify the integrin profile of isolated human upper airway basal cells. These basal cells could be maintained on type I collagen for at least 7 days, where they became partially confluent and retained expression of cytokeratins 5 and 14. Availability of pure populations of basal cells should permit investigations of their role in both normal and maladaptive repair of adult upper airway epithelium.     

Left-right patterning in the mouse requires Epb4.1l5-dependent morphogenesis of the node and midline

The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.     

The Idiopathic Pulmonary Fibrosis Honeycomb Cyst Contains A Mucocilary Pseudostratified Epithelium

Background

We previously identified a MUC5B gene promoter-variant that is a risk allele for sporadic and familial Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP). This allele was strongly associated with increased MUC5B gene expression in lung tissue from unaffected subjects. Despite the strong association of this airway epithelial marker with disease, little is known of mucin expressing structures or of airway involvement in IPF/UIP.

Methods

Immunofluorescence was used to subtype mucus cells according to MUC5B and MUC5AC expression and to identify ciliated, basal, and alveolar type II (ATII) cells in tissue sections from control and IPF/UIP subjects. Staining patterns were quantified for distal airways (Control and IPF/UIP) and in honeycomb cysts (HC).

Results

MUC5B-expressing cells (EC) were detected in the majority of control distal airways. MUC5AC-EC were identified in half of these airways and only in airways that contained MUC5B-EC. The frequency of MUC5B+ and MUC5AC+ distal airways was increased in IPF/UIP subjects. MUC5B-EC were the dominant mucus cell type in the HC epithelium. The distal airway epithelium from control and IPF/UIP subjects and HC was populated by basal and ciliated cells. Most honeycombing regions were distinct from ATII hyperplasic regions. ATII cells were undetectable in the overwhelming majority of HC.

Conclusions

The distal airway contains a pseudostratified mucocilary epithelium that is defined by basal epithelial cells and mucus cells that express MUC5B predominantly. These data suggest that the HC is derived from the distal airway.