Jason R. Rock

Basal cells as stem cells of the mouse trachea and human airway epithelium

The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreERT2 transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.

Distinct stem cells contribute to mammary gland development and maintenance

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling

The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed ‘pathological airway remodeling’, affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6–30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.

Epithelial Progenitor Cells in Lung Development, Maintenance, Repair, and Disease

The vertebrate lung is elegantly patterned to carry out gas exchange and host defense. Similar to other organ systems, endogenous stem and progenitor cells fuel the organogenesis of the lung and maintain homeostasis in the face of normal wear and tear. In the context of acute injury, these progenitor populations are capable of effecting efficient repair. However, chronic injury, inflammation, and immune rejection frequently result in pathological airway remodeling and serious impairment of lung function. Here, we review the development, maintenance, and repair of the vertebrate respiratory system with an emphasis on the roles of epithelial stem and progenitor cells. We discuss what is currently known about their identities, lineage relationships, and the mechanisms that regulate their differentiation along various lineages. A deeper understanding of these progenitor populations will undoubtedly accelerate the discovery of improved cellular, genetic, molecular, and bioengineered therapies for lung disease.

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca2+‐activated Cl− channels (CaCC) and non‐selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non‐human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4′‐diisothiocyano‐2,2′‐stillbene‐disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration‐dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16atm1Bdh/tm1Bdh) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.

Notch-Dependent Differentiation of Adult Airway Basal Stem Cells

The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily toward secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways.

Studies on expression and function of the TMEM16A calcium-activated chloride channel

Calcium-activated chloride channels (CaCC) with similar hallmark features are present in many cell types and mediate important physiological functions including epithelial secretion, sensory signal transduction, and smooth muscle contraction. Having identified TMEM16A of the transmembrane proteins with unknown function (TMEM) 16 family as a CaCC subunit, we have developed antibodies specific for mouse TMEM16A, as evidenced by the absence of immunoreactivity in TMEM16A knockout mice. Here, we show that TMEM16A is located in the apical membranes of epithelial cells in exocrine glands and trachea. In addition, TMEM16A is expressed in airway smooth muscle cells and the smooth muscle cells of reproductive tracts, the oviduct and ductus epididymis. In the gastrointestinal (GI) tract, TMEM16A is absent from smooth muscle cells, but present in the interstitial cells of Cajal (ICC), the pacemaker cells that control smooth muscle contraction. The physiological importance of TMEM16A is underscored by the diminished rhythmic contraction of gastric smooth muscle from TMEM16A knockout mice. The TMEM16A expression pattern established in this study thus provides a roadmap for the analyses of physiological functions of calcium-activated chloride channels that contain TMEM16A subunits.

Transmembrane Protein 16A (TMEM16A) Is a Ca2+-regulated Cl– Secretory Channel in Mouse Airways

For almost two decades, it has been postulated that calcium-activated Cl– channels (CaCCs) play a role in airway epithelial Cl– secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a–/–) to investigate the role of this channel in Cl– secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a–/– tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family, including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl– secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl– secretion reflected ∼50% CFTR activity and ∼50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a–/– and the CFTR–/– mice exhibited similar congenital cartilaginous defects that may reflect a common Cl– secretory defect mediated by the molecularly distinct Cl– channels. Importantly, the residual CaCC activity in Tmem16a–/– mice appeared inadequate for normal airway hydration because Tmem16a–/– tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl– secretion appears to be necessary for normal airway surface liquid homeostasis.

The transmembrane protein TMEM16A is required for normal development of the murine trachea

Pathological collapsibility of the upper airways, caused by many different genetic and environmental insults, is known as tracheomalacia in humans. We determined that Tmem16a, a member of an evolutionarily conserved family of predicted transmembrane proteins, is expressed in the developing trachea. We report that all mice homozygous for a null allele of Tmem16a died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea. In addition, the development of the trachealis muscle that spans the dorsal aspect of the trachea was abnormal in Tmem16a mutants. Since the chondrogenic mesenchyme does not express Tmem16a at any time, we propose that the cartilage ring defect observed in Tmem16a mutants is secondary to an expansion of the embryonic trachea that might result from improper stratification of the embryonic tracheal epithelium or the abnormal trachealis muscle. Our data identify Tmem16a as a novel regulator of epithelial and smooth muscle cell organization in murine development. This mutant, the first knockout of a vertebrate TMEM16 family member, provides a mouse model of tracheomalacia.

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.