Observing planar cell polarity in multiciliated mouse airway epithelial cells

Abstract

The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type.

Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

“…A MOC of 1.00 indicates completely uniform Vangl1 localization, whereas maximal asymmetric localization produces a MOC of approximately 0.5 (see Methods for details). Crescent localization of core PCP proteins can also be observed in primary mouse tracheal epithelial cell (MTEC) cultures generated from isolated basal cells that have been differentiated at an air-liquid interface (ALI, Figure 1) (22, 23). Despite the absence of a tissue axis to globally organize polarization in MTECs, crescents (and cilium polarity) are coordinated among groups of neighboring cells by the activity of the core PCP signaling mechanism (9).…”

“…Airway clearance is known to be disrupted in CF, CRS, and many other diseased airway epithelia (1), so we assessed motile cilium polarity by transmission electron microscopy (TEM). The orientation of the basal foot ciliary appendage points toward ciliary beat direction, and a line drawn through the axonemal central microtubule doublet is perpendicular to ciliary beat direction (23) (Supplemental Figure 5A). We obtained healthy, CF, and CRS sinonasal tissues as above, and first assessed VANGL1 localization (see Figure 4A) and the presence and morphology of MCCs by antibody labeling and scanning electron microscopy (SEM) for normal and CF (Supplemental Figure 5B, CRS not shown).…”

“…We have previously shown that motile cilia are oriented by PCP signaling, and that localization of Vangl1 and other core signaling components to the P-D side apical junctions of airway epithelial cells reports the activity of this pathway (9, 23). We examined the kinetics of core PCP protein (Vangl1) localization in the mouse airway epithelium, and found that PCP emerges in a 2-phase process.…”

Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity

“…In the trachea, motile cilia adorn the surface of epithelial cells and are uniformly oriented, which drives a directional outward flow that is critical for mucociliary clearance232627. In the brain ventricles, motile cilia are also uniformly oriented to drive a directional cerebrospinal fluid flow that provides directional cues for brain development and is required for normal spine curvature2328293031.…”

The core planar cell polarity gene,Vangl2, directs adult corneal epithelial cell alignment and migration

“…PCP activity has been shown to modulate cancer cell invasion metastasis, but tumours themselves fundamentally recapitulate ‘embryonic’ systems [32]. It seems very likely that any epithelial system that has the capacity to repair and regenerate in the adult may use a PCP-based mechanism: postnatal airway epithelial cells display robust planar polarity, and fish lateral lines are another example of potential persistence of PCP into the adult [33,34]. However, there is little experimental evidence to elucidate the mechanisms of action.…”