In different organs, epithelial cells characteristically show apical–basal polarity, which is necessary for their function as barriers between different extracellular environments (Drubin and Nelson, 1996
; Mostov et al., 2000
). It is well established that actin and actin-associated proteins are essential for generating molecular and morphological cell polarity (Winder and Ayscough, 2005
). In distal lung epithelium, as in other epithelial cell types, f-actin myosin II-b, β-catenin and zonula occludens protein (ZO-1) localize predominantly to the apical junctional complex, which is essential for cell polarization (Tepass, 2003
; , and data not shown). Similar asymmetric apical localization has been seen for E-cadherin as well as for pericentrin, a conserved centrosome protein involved in microtubule organization that is located apically in epithelial cells (; Doxsey et al. 1994
; Chenn et al. 1998
), and also for polarity proteins Par-3 and Par-6 (). These data suggest that distal embryonic lung epithelial cells are indeed polarized.
Lung distal epithelium is polarized
We next addressed whether distal embryonic lung epithelial cells divide perpendicular to the basement membrane or laterally within the plane of the epithelium. At the pseudoglandular stage, staining with tubulin, an indicator of cells in metaphase (Daar et al., 1991
), and PCNA antibodies showed that distal epithelial cells are indeed highly proliferative/mitotic (). Most of these mitotic cells were apparently dividing perpendicularly to the basement membrane, as judged by staining with anti-tubulin, which localized at apical and basal sides of mitotic cells (; arrowheads).
Lung distal epithelium is mitotic with perpendicular cell divisions
To facilitate quantification of cells dividing perpendicularly versus laterally, we first stained distal epithelium for centrosomes with pericentrin antibody. Then, mitotic cells were quantified based on centrosome orientation relative to the collagen IV-stained basement membrane, in order to distinguish parallel (lateral) from perpendicular spindle alignments in mitotic cells (, ). Centrosomes that were oriented at 0 ± 30° to the basement membrane were classed as parallel; those that were oriented at 90 ± 30° were classed as perpendicular. In E14-14.5 distal epithelium, most cell divisions (82%) occurred perpendicular to the basement membrane, while the remaining 18% of mitotic cells had an alignment that appeared parallel (, 82.0 ± 3.0% vs. 18.0 ± 2.0%, respectively; n = 3).
The regulation of the spindle orientation and cell division axis, which can impact normal physiological processes, including epithelial tissue branching and differentiation is often associated with cell polarity regulation in polarized cells in model organisms (reviewed in Betschinger and Knoblich, 2004
). The orientation and positioning of the mitotic spindle, which determines the plane of cell division, is tightly regulated in polarized cells such as epithelial cells by intrinsic and extrinsic cues. These cues include certain cell polarity regulating asymmetrically localized cortical proteins, cell–cell adhesion, and cell-matrix adhesion. Therefore, to further distinguish parallel from perpendicular spindle alignments, and to distinguish how distal epithelial cells control their spindle orientation, we next determined the expression pattern of asymmetrically localized cortical proteins, which have spindle orientation-regulatory functions ().
Expression of the polarity proteins LGN, mInsc, NuMA and Numb in lung distal epithelium
In Drosophila, chick and mammalian epithelium, a protein complex containing Inscuteable, LGN and NuMA captures a spindle pole at the apical cortex, aligning the spindle with the apical–basal axis and thus regulating cell fate (Kraut et al., 1996
; Du et al., 2001
; Roegiers and Jan, 2004
; Lechler and Fuchs, 2005
; Žigman et al., 2005
; Morin et al., 2007
). Immunofluorescence and Western blot showed that the expression of mouse Inscuteable (mInsc), LGN, NuMA and the cell fate determinant Numb is conserved in distal lung epithelium (). In the mammalian epithelium, LGN, NuMA and mInsc proteins localize to the apical side of cells only during apical-basal (perpendicular) cell division, and have a diffuse localization in cells that are in interphase or that are undergoing lateral divisions (Lechler and Fuchs, 2005
). As expected, apical localization of LGN, mInsc and NuMA was seen at the cortex of most mitotic cells, and accounted for more than 80% of all mitoses in distal embryonic lung epithelial cells (; n
= 3), which further suggests that most of these cells divide perpendicularly.
In epithelial and other cell types, the axis of polarity that will determine the orientation of the apical-basal cell division plane is defined by cell fate determinants (CFDs), such as Numb and Par-proteins. Intrinsic CFDs are asymmetrically localized in dividing cells, and preferentially segregate into one of two sibling daughter cells in order to mediate asymmetric cell divisions (Betschinger and Knoblich, 2004
). We found that the cell fate determinant Numb had a polarized asymmetric localization at the apical side of distal epithelial cells with little or no staining at the basal pole (). Closer inspection of mitotic cells revealed that Numb staining is consistently concentrated as a crescent at the apical pole of one daughter cell (apical daughter) in 88± 5.0% of wildtype distal epithelial tip cells (, n
= 3). Thus, the more perpendicular a cell division is (, ), the more likely it is to segregate Numb preferentially to one daughter cell in mitotic lung epithelial cells.
In this brief study, to our knowledge, we provide the first evidence that embryonic distal lung epithelium is polarized with characteristically perpendicular divisions. We also demonstrate for the first time that the expression of the polarity proteins mInsc, LGN and NuMA, which control mitotic spindle orientation (Siller and Doe, 2009
; Zheng et al., 2010
), are conserved in embryonic distal lung epithelial cells. In addition, we show that the cell fate determinant Numb is asymmetrically localized/segregated in mitotic distal epithelium
Alveolar hypoplasia, which characterizes bronchopulmonary dysplasia in extremely premature human infants, wherein a significant deficiency of stem/ progenitor cells probably occurs, are common features of human prematurity and/or lung injury and are thus of major public health concern. Cell polarity is critical for the proper balance between self-renewal and differentiation of lung specific progenitors, which is absolutely required for normal lung morphogenesis and regeneration. Moreover, loss of epithelial cell polarity is involved in several lung diseases, including lung epithelial cancers and Chronic Obstructive Pulmonary Disease, which are likewise related to disruption of lung epithelial differentiation and cellular function (Xu et al., 2006
). Therefore, understanding the behavior of lung epithelial progenitors localized in the distal embryonic lung epithelium, as described herein, could identify innovative solutions to restoring normal lung morphogenesis and possibly regeneration of the gas diffusion surface. Our findings now provide a framework for future translationally orientated studies in this area.