Deletion of Sox2 from Clara cells in the postnatal bronchiolar epithelium caused the progressive loss of ciliated, Clara, and goblet cells and an inability to produce goblet cells in response to allergen challenge. Taken together, expression of Sox2 in Clara cells is required for differentiation and/or maintenance of ciliated, Clara, and goblet cells in bronchiolar epithelium after birth. The findings support the concept that Clara cells serve as the common progenitors of ciliated, Clara, and goblet cells in a process requiring Sox2. Remarkably, Sox2Δ/Δ mice survive normally in the vivarium in spite of the widespread loss of ciliated, Clara, and goblet cells from conducting airways.
The Clara cell of the intrapulmonary airway is capable of self-renewal and transdifferentiation into ciliated and goblet cells 
, and has been termed a facultative progenitor in lung homeostasis 
. In the present study, we utilized the rat Scgb1a1
promoter that is specifically active in Clara but not in ciliated or neuroepithelial cells. The loss of Clara, ciliated and goblet cells in response to Sox2
deletion are consistent with the proposed role of Clara cells as progenitor cells in bronchioles, and show that Sox2
is necessary for normal proliferation and differentiation of the major epithelial cell types lining the bronchioles ().
Model in which Sox2 is required for Clara cell self renewal and differentiation of facultative progenitors of the bronchiolar epithelium.
Experiments observing the steady-state pattern of chimeric labeled mouse embryonic stem cells 
, and pulse-lineage-labeled newborn Clara cells 
show that for homeostasis of the intrapulmonary respiratory epithelium, most progenitor cells (e.g. Clara cells) are randomly distributed throughout the bronchioles and are not associated with particular anatomical niches. Interestingly, unlike bronchiolar Clara cells, numbers of tracheal Clara cells that were labeled with a conditional Cre-ER in the newborn period, steadily declined as the mice aged. Thus, progenitor cell activity in the trachea may be somewhat different than that in the bronchioles 
, findings consistent with previous studies demonstrating the importance of tracheal basal cells as progenitors during repair 
. Epithelial cells in the submucosal glands and basal cells in larger airways are able to contribute to epithelial repair. Although recent studies demonstrate that progenitor cells in rare anatomical niches were not responsible for bronchiolar maintenance, there is evidence that they play a role in lung repair after extensive injury 
. Bronchio-alveolar stem cells (BASCs) have been proposed as a uniquely proliferative stem cell 
, although recent evidence supports the concept that Clara cells or toxicant resistant Clara cells are the primary progenitors during repair of the postnatal lung airway 
The present study and recent findings, in which Sox2
was deleted in the embryonic lung and trachea, support the concept that progenitor cell relationships are somewhat distinct in the proximal and peripheral airways. An Nkx2.5-Cre
construct was used recently to delete Sox2
from the ventral epithelial domain of the anterior foregut prior to lung budding and the consequences on tracheal development were examined 
. The deletion of Sox2
occurred earlier than in the present model and resulted in abnormal laryngeal/tracheal cartilage formation and perinatal death. In that study, a small subset of tracheal cells lacked markers of differentiation and numbers of tracheal Clara and ciliated cells were reduced. In contrast to findings in the present study, increased numbers of tracheal goblet cells were observed and tracheal proliferation was normal following the early deletion of Sox2
from the embryo 
. In the present study, Sox2
was deleted in the perinatal/postnatal period, after lung branching morphogenesis was complete, and its consequences on postnatal growth and allergen challenge were examined. Perinatal deletion of Sox2
resulted in progressive loss of both ciliated and Clara cells in the bronchiolar epithelium. In response to the allergen, goblet cell differentiation was absent in the Scgb1a1-Cre Sox2Δ/Δ
mice in spite of extensive inflammation. Goblet cells were absent at baseline and after allergen challenge as indicated by mucin, Muc5AC, and Spdef staining in the bronchioles of the Scgb1a1-Cre Sox2Δ/Δ
mice. The significant temporal and spatial differences in which Sox2
was deleted are likely responsible for the differences in goblet cell differentiation seen in these two systems.
A tamoxifen-inducible CMV-CreER
construct was used recently to delete Sox2
in adult mice and its effect on tracheal homeostasis studied 
. In the adult tracheal epithelium, reduced cell proliferation and cell height and increased cell width were observed. In the trachea, numbers of Clara cells were not reduced after deletion of Sox2
, perhaps indicating the importance of basal cells to the homeostasis of pseudostratified epithelium of the proximal airway. Recent lineage analysis of Clara cells in the trachea and bronchioles support their role in bronchiolar homeostasis, but not in the trachea where other cell types, presumably basal cells, play an important role.
Clara cells are a source of bronchiolar ciliated cells after birth 
, and ciliated cells are long-lived 
. In the present mouse model, the progressive loss of Clara cells occured during the rapid growth phase (0–4 wks) and was faster than loss of ciliated cells, as indicated by the expression of FoxJ1 and α-tubulin. The lack of cleaved Caspase 3 staining after deletion of Sox2
suggests that the loss of cell number is not related to apoptosis. Thus it is likely that the progressive loss of the columnar morphology is mediated by the decreased proliferation, the gradual loss of ciliated cells that were differentiated before the deletion of Sox2
in their progenitors (Clara cells) and the rapid loss of differentiated features of Clara cells after deletion of Sox2
. These findings suggest that Sox2 plays a role in Clara cell differentiation, proliferation and progenitor cell capacity.
The deletion of Sox2
from the bronchial epithelium did not influence the expression of Nkx2.1 or Foxa2, transcription factors important for the expression of differentiation markers. Thus the loss of the Clara cell marker Scgb1a1 (a direct target of both Nkx2.1 and Foxa2) is not caused by the loss of Nkx2.1 or FoxA2. In the present study, in vitro
promoter assays demonstrated that Sox2 was not a synergistic partner of Nkx2.1 or FoxA2 in the activation of Scgb1a1
. The ubiquitous presence of Sox2 in normal Clara, ciliated, and goblet cells, and the activation of the Scgb1a1
, and Agr2
promoter-luciferase constructs by Sox2 support the role of Sox2 as a positive regulator of these differentiation markers. Taken together, the results demonstrate that Sox2 is necessary for differentiation and maintenance of Clara, ciliated, and goblet cells from Clara cell progenitors, and may play a role in directly regulating the expression of differentiation dependent genes in each of these cell types. Recent studies demonstrated the loss of both Nkx2.1 and FoxA2 during allergen induced differentiation of goblet cells 
. The lack of goblet cells during allergen challenge to Sox2Δ/Δ
mice supports the concept that Clara cell differentiation, dependent upon Sox2, is required for the differentiation of Clara cells into goblet cells following allergen exposure. The bronchiolar epithelium of the Sox2Δ/Δ
mice lacked both Muc5AC and Spdef (). Perhaps consistent with the requirement for Sox2 in goblet cell differentiation, Sox2 activated a Muc5AC
-luciferase reporter in colorectal tumors 
, and Muc5B
mRNA was increased in lung when Sox2 was overexpressed in a transgenic mouse model 
In the present study, cell proliferation was significantly reduced in the bronchioles after perinatal deletion of Sox2
using a Scgb1a1-Cre
transgene, a finding consistent with reduced cell proliferation seen in the adult trachea after deletion of Sox2
from mature trachea using an CMV-CreER
. While mechanisms controlling its role in bronchiolar cell proliferation are unclear at present, Sox2 promotes proliferation in breast cancer cell lines 
and the loss of Sox2
promotes terminal differentiation of neuronal stem cells 
. The functions of Sox2 are highly dependent on cell types and developmental cassettes. Sox2 mediates cellular reprogramming required for pluripotent stem cell activity in fibroblasts. Sox2 interacts with multiple transcriptional partners and co-activators that are influenced by developmental, cellular, and tissue dependent factors. TGF-β/Smad3 signaling is a potent anti-proliferative pathway in multiple epithelial types, including respiratory epithelial cells, and promotes squamous differentiation of bronchiolar epithelial cells in vitro 
. In the present study, we observed that Sox2, like Sox17, bound to Smad3 and inhibited its activity, providing a potential mechanism by which Sox2 may influence cell proliferation and differentiation.
In summary, the expression of Sox2 in Clara cells of the bronchiolar epithelium was required for its normal differentiation and proliferation after birth. The present data demonstrate the importance of both Sox2 and Clara cells (the latter serving as facultative progenitors of Clara, ciliated, and goblet cells) in proximal airway differentiation and homeostasis. Sox2 was required for the expression of Spdef, a gene required for postnatal goblet cell differentiation and mucus hyperproduction in response to pulmonary allergen sensitization. In spite of the extensive loss of Clara, ciliated, and goblet cells, mice in which Sox2 was extensively deleted from the respiratory epithelium do not develop spontaneous infections and survive normally in the vivarium, indicating an unexpected compensatory capacity in the maintenance of homeostasis in spite of extensive changes to the structure of the conducting airway.