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Key Event Title
Motile Cilia Number/Length, Decreased
|Level of Biological Organization|
|multi-ciliated epithelial cell|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|ox stress-mediated FOXJ1/cilia/CBF/MCC impairment||KeyEvent||Karsta Luettich (send email)||Open for comment. Do not cite|
|During development and at adulthood||High|
Key Event Description
Motile cilia are microtubule-based organelles that protrude from the cell surface and generate directional flow of fluid with coordinated beating. 50% to 80% of human respiratory epithelium is comprised of ciliated cells covered with multiple motile cilia that move mucus (together with mucus-trapped substances) upward for clearing the airways (Yaghi and Dolovich, 2016). The ciliated airway epithelial cells are typically covered by more than hundred motile cilia (Bustamante-Marin and Ostrowski, 2017). On average, 150 motile cilia were counted per ciliated human epithelial cell in the study by Mao et al. (Mao et al., 2018). In an earlier report, 200 motile cilia per ciliated cell in human trachea is mentioned (Wanner et al., 1996), and, in a more recent study, a range of 100 to 600 ciliary precursors were counted in fully differentiated mouse tracheal epithelial cells correlated with increasing surface area (Nanjundappa et al., 2019). Cilia are 6–7 µm long and 0.2–0.3 µm in diameter (Brooks and Wallingford, 2014; Yaghi and Dolovich, 2016). Ciliated cell density and the motile cilia length and number per cell correlate with ciliary beating frequency which is routinely used as a predictor of the mucociliary clearance efficiency (King, 2006). Morphological changes of airway cilia are expected to impact multiple motile cilia functional integrity. This key event represents the decrease in the numbers or absence of motile cilia or reduction in length of motile cilia.
How It Is Measured or Detected
Acetylated tubulin is a common ciliary marker (Kim et al., 2013; Piperno and Fuller, 1985), and apical acetylated tubulin staining with subsequent microscope image scoring is a frequently used method of cilia detection and enumeration (Johnson et al., 2018; Mao et al., 2018; Stubbs et al., 2008). Staining of beta-tubulin IV, a protein enriched in motile cilia, is another common method of cilia detection (Brekman et al., 2014; Milara et al., 2012). Ciliated cells can also be identified by the presence of axonemal structures on the cell surface using scanning electron microscopy (Gomperts et al., 2007). Mature cilia numbers could be deduced from ciliary precursors in immunofluorescence assays: ciliary precursors can be calculated from three-dimensional superresolution structured illumination microscopy (3D-SIM) images using e.g. a spot detection tool (Nikon Elements AR 4 Software) (Nanjundappa et al., 2019).
For cilia length measurement, the ciliated cells/tissue needs to be stained (Diff-Quik: Dade Behring stain, hematoxylin and eosin staining, labelling with antibodies for ciliary markers such as alpha-tubulin), visualized by microscopy and cilia length quantified (using e.g. ImageJ software or MetaMorph Microscopy Automation & Image Analysis Software) (Brekman et al., 2014; Leopold et al., 2009b; Li et al., 2014). Generally, multiple measurements of one sample and multiple sample preparations of cells/tissues are imaged for reliable quantitation.
Domain of Applicability
The ultrastructural features of human and other mammalian respiratory epithelial cilia and those from lower animals (e.g. flatworms and mollusks) are remarkably similar (Meunier and Azimzadeh, 2016; Wanner et al., 1996). The master regulators of multiciliated cell differentiation, such as NOTCH, GEMC1, MCIDAS, FOXJ1, RFX2/3 are conserved throughout vertebrates (e.g. mammals, Xenopus, zebrafish) and multiple motile cilia across these organisms are functionally similar in generating fluid flow through coordinated beating (Choksi et al., 2014; Meunier and Azimzadeh, 2016; Wessely and Obara, 2008).
The motile cilia numbers reach adult levels in the mouse airway epithelium at day 21 after birth (Rawlins et al., 2007; Toskala et al., 2005). At birth, there is no discernable cilia-generated airway fluid flow in mice (Francis et al., 2009). Between postnatal days 3 and 7 the flow is established in trachea correlating with the increase in the density of ciliated cells in the tracheal epithelia (Francis et al., 2009). After airway fluid flow establishment, the KE is applicable to all life stages.
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Brooks, E.R. and Wallingford, J.B. (2014). Multiciliated cells. Curr. Biol. 24, R973-982.
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Mao, S., Shah, A.S., Moninger, T.O., Ostedgaard, L.S., Lu, L., Tang, X.X., et al. (2018). Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling. Proc. Natl. Acad. Sci. U. S. A. 115, 1370-1375.
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Milara, J., Armengot, M., Bañuls, P., Tenor, H., Beume, R., Artigues, E., et al. (2012). Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro. Brit. J. Pharmacol. 166, 2243-2262.
Nanjundappa, R., Kong, D., Shim, K., Stearns, T., Brody, S.L., Loncarek, J., et al. (2019). Regulation of cilia abundance in multiciliated cells. Elife 8, e44039.
Piperno, G. and Fuller, M.T. (1985). Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085-2094.
Rawlins, E.L., Ostrowski, L.E., Randell, S.H. and Hogan, B.L. (2007). Lung development and repair: contribution of the ciliated lineage. Proc. Natl. Acad. Sci. U. S. A. 104, 410-417.
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