Temporal and spatial distribution of ciliogenesis in the tracheobronchial airways of mice

Abstract

Little is known about ciliogenesis as it proceeds through the entire airway tree, from the trachea to the terminal bronchioles, especially during the postnatal period. The purpose of this study was to define the spatial and temporal (prenatal and postnatal) pattern of normal cilia development in the mouse. Three airway generations representing the entire airway tree were examined: trachea, lobar bronchi, and terminal bronchiole. Ciliated cells in lung lobe whole mounts were labeled with a fluorescent dye for confocal microscopy, and ciliated cell surface density was measured for each airway generation and age. The same samples were examined by scanning electron microscopy to verify the appearance of ciliated cells among the differentiating epithelium of the airways. Ciliated cells were first detected in the trachea and lobar bronchi at 16 days gestational age (DGA) and in the terminal bronchioles at 18 DGA. Ciliated cell surface density increased with prenatal and postnatal age at all airway levels. However, the ciliated cell surface density of the trachea and lobar bronchi was always greater compared with the terminal bronchiole. In conclusion, the study revealed that in developing tracheobronchial airways of the mouse: 1) Ciliogenesis differs temporally and spatially by airway generation; 2) Ciliated cell surface density increases with age in all airway generations, but density decreases in a proximal to distal direction; and 3) A significant portion of ciliogenesis continues after birth. This study provides a healthy basis for investigations of neonatal pulmonary disease or pollutant toxicity affecting cilia and its functions.

Figure 1

Microscopic images of adult mouse trachea labeled with FITC conjugated tomato lectin taken with (A) scanning confocal microscope (B) epifluorescence (C) transmitted light. Ciliated areas shown with epifluorescence are the same areas observed with transmitted light. Bar = 10 micrometers.

Figure 2

Comparison of age-related ciliated cell surface density in three airways of mouse lung: (A) trachea (B) lobar bronchi (C) terminal bronchioles. Time points include 16, 17, 18, and 19 days gestation (DGA); 1, 7, 14, and 21 days postnatal (DPN); and adult. Ciliated cell surface density is expressed as percentage of airway surface area. NS = No significant differences within each airway level between time points in brackets. Otherwise, the significance within each airway level between time points is p < 0.05. * = significantly different (p < 0.05) from the trachea and lobar bronchi at the same time point. + = significantly different (p < 0.05) from the other two airway generations at the same time point.

Figure 3

SEM images of the development of cilia in the trachea of mice at different ages: 16 DGA (A, B), 19 DGA (C, D), 14 DPN (E, F), and 21 DPN (G, H). At 16 DGA, sparse, single ciliated cells were visible. The remaining cells were undifferentiated and round, lacking microvilli or apical projections. At 19 DGA, cilia were short and covered the surface of ciliated cells. Cilia cells appeared alone or in pairs. Nonciliated cells were flat and had some microvilli. At 14 DPN, ciliated cells were covered by long cilia and occurred in pairs or in rows of 3–4 cells. At 21 DPN, ciliated cells have long cilia. Bar = 10 micrometers in A, C, E, and G. Bar = 50 micrometers in B, D, F, and H.

Figure 4

SEM images of the development of cilia in the terminal bronchiole. (A) Ciliated cells were first seen at 18 DGA and were single, fairly flat cells with very short cilia and microvilli. Nonciliated cells surrounding the ciliated cells were undifferentiated and slightly raised. (B) At 14 DPN, ciliated cells had long cilia and were alone or in pairs. Bar = 10 micrometers in A and B. Arrows indicate cilated cells.

Figure 5

SEM images of (A) trachea (B) lobar bronchi and (C) terminal bronchiole in adult mice. Cilia are long and cover the entire surface of the cell and extend over the cell margins in the trachea and lobar bronchi. Ciliated cells appeared in rows of 4–6 cells. In the terminal bronchioles, cilia are shorter and cells appear in single or in pairs. Bar = 10 micrometers.