Cell polarity and spindle orientation in the distal epithelium of embryonic lung

Abstract

A proper balance between self-renewal and differentiation of lung-specific progenitors at the distal epithelial tips is absolutely required for normal lung morphogenesis. Cell polarity and mitotic spindle orientation play a critical role in the self-renewal/differentiation of epithelial cells and can impact normal physiological processes, including epithelial tissue branching and differentiation. Therefore, understanding the behavior of lung distal epithelial progenitors could identify innovative solutions to restoring normal lung morphogenesis. Yet little is known about cell polarity, spindle orientation, and segregation of cell fate determinant in the embryonic lung epithelium, which contains progenitor cells. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized and highly mitotic with characteristic perpendicular cell divisions. Consistent with these findings, mInsc, LGN, and NuMA polarity proteins, which control spindle orientation, are asymmetrically localized in mitotic distal epithelial progenitors of embryonic lungs. Furthermore, the cell fate determinant Numb is asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. These findings provide evidence for polarity in distal epithelial progenitors of embryonic lungs and provide a framework for future translationally oriented studies in this area.

Figure 1. Lung distal epithelium is polarized

Immunofluorescence at E14 shows strong signals for actin (A), myosin II-b (B), pericentrin (C), E-cadherin (D), Par-3 (E) and Par-6 (F) proteins at the apical side of distal epithelial cells (arrowheads). Dashed line represents the collagen IV-stained basement membrane. Insets in C,D represent the area marked with an asterisk in the same panel. Inset in F shows immunofluorescence staining of E14 basement membrane (arrows) with collagen IV antibody. Scale bars: 50 μm.

Figure 2. Lung distal epithelium is mitotic with perpendicular cell divisions

(A-D) Antibody staining at E14 shows strong signals for α-tubulin and PCNA in distal (arrowheads) rather than proximal (arrow; A) epithelium. (D) represents electronic magnification of boxed area in C, and shows perpendicular/apical-basal (arrowheads) spindle alignments relative to collagen IV-stained basement membrane (dashed line) in mitotic cells (a,b,c). (E-F) Most mitotic cells in the distal epithelium divide perpendicularly, as represented by the perpendicular orientation of pericentrin-stained centrosomes (arrowheads, arrows) relative to the basement membrane (dashed line; E,F), while only a few mitotic cells have their centrosomes aligned parallel to the basement membrane (G; arrowheads). (G) Quantitation of spindle orientations, expressed as a percentage of all divisions in the distal epithelium from the experiments shown in E-F (n=48). Scale bars: 50 μm.

Figure 3. Expression of the polarity proteins LGN, mInsc, NuMA and Numb in lung distal epithelium

as shown by immunofluorescence (A-D) and Western blot (E). Note polarized apical localization of these proteins (A-D; arrowheads) relative to collagen IV-stained basement membrane (dashed line). (F) Quantitation of the apical localization of proteins shown in A-C, which is expressed as a percentage of all cells in the distal epithelium (n=87). (G) Quantification of late mitotic distal epithelial cells, with Numb inherited by one (inset in G, which represents the area marked with an asterisk in D) or both daughter cells at E14. This is expressed as a percentage of all divisions in the distal epithelium (n=42). Scale bars: 50 μm.