Ontogeny and regulation of the airway stem cell pool in the lung.
Tissue-specific stem cells are crucial in maintaining homeostasis and integrity of adult organs. In the respiratory tract this function has been attributed largely to basal cells, multipotent epithelial progenitors identified largely by expression of p63 and intermediate filament keratins. Little is known about how these populations arise during development and how they acquire their molecular features and properties as tissue specific stem cells of the adult lung. We are addressing these issues and have shown the appearance of p63+ cells in lung progenitors unexpectedly at very early developmental stages. We found that they undergo major lineage-restriction events and showed their key contribution to the adult basal cell pool, including a rare subpopulation that is the source of the aberrant alveolar remodeling by H1N1 viral infection (Yang et al. Dev Cell 2018). We are studying the molecular mechanisms underlying these events and basal cell plasticity.
Our studies provide evidence that the stem cell pool also includes a population of uncommitted p63-negative cells that occupies a parabasal position in the airway epithelium. These parabasal cells activate Notch3 to function as a gate keeper controlling expansion of the basal cells, and are markedly decreased in human patients with chronic pulmonary diseases. Our studies suggest the parabasal-basal cell imbalance to be a hallmark of airways from COPD patients (Mori et al. Development 2015). We are exploring these mechanisms in mouse genetic models and in human studies.
Developmental signaling in lung formation and in diseases.
We have a long-lasting interest on elucidating how specific gene regulatory networks control formation of the lung primordium, airway branching and differentiation and ultimately are implicated in birth defects and neonatal conditions. These studies have revealed key roles for Fgf, Tgfb, Wnt, retinoids, matrix components and the Hippo pathway in this regulatory network (vanSoldt et al. Development 2019, Qiu et al. Nature Methods 2018, Chen et al. J Clin Invest 2010, Cardoso & Lu Development 2006).
Lately we uncovered an unsuspected role for Yap and its nucleocytoplasmic shuttling in the establishment of proximal-distal cell fates, morphogenesis and epithelial integrity in the developing lung (vanSoldt et al. Development 2019, Mahoney et al. Dev Cell 2014). We showed that Yap signaling is required to form airway epithelial progenitors and prevent aberrant cystic expansion of the distal lung. However maintaining nuclear Yap active in developing airways markedly alters differentiation inducing ectopic alveolar cell fate in airways. We are investigating mechanisms of cell plasticity and molecular determinants of cell fate when airways are forming using unbiased single-cell transcriptomics and epigenomic approaches. We have recently integrated these concepts on developmental signaling with stem cell technology in innovative studies that first successfully generated functional lungs in vivo by blastocyst complementation (Mori et al Nature Med, 2019)
We also study how the various epithelial cell types of the mature airways (secretory, multiciliated, neuroendocrine) arise from undifferentiated progenitors and how their fates are balanced in developing and adult airways. These studies provide insights into the origin and consequences of the aberrant differentiation programs in pulmonary disease pathogenesis. For example, we first demonstrated that secretory cells do not form in the absence of Notch signaling and that, postnatally, Notch continues to be required to prevent aberrant secretory differentiation and mucous metaplasia (Tsao et al. Development 2009, 2011, PNAS 2016). We showed the impact of Notch in neuroendocrine (NE) cell fate and recently provided novel evidence of three distinct populations of NE cells and their selective contribution to NE hyperplasias in human diseases (Mou et al. Cell Rep. 2021, Guha et al. PNAS 2012).
Our studies also led to the identification of an UpK3a-expressing population of progenitor cells that contributes to regenerate the airway epithelium post injury. We found these progenitors present in the human lungs and aberrantly expanded in NE hyperplasias of infants and adults (Guha et al. Cell Rep 2017). We have recently established new technologies for isolation and characterization of human upper airway progenitors to investigate and model diseases of prematurity in human neonates (Shui et al. Sci Rep 2021). Lastly, we are using cutting-edge cellular, single-cell transcriptomics and computational approaches to study SARS-COVID-2-host interactions and identify druggable targets in the human lung.
Postnatal consequences of prenatal disruption of developmental signaling
Developmental defects, such as tracheoesophageal fistula, pulmonary hypoplasia and failure to form the lungs are known for decades to be part of the "Vitamin A deficiency syndrome." We have been studying the pathogenesis of these defects and identified a gene network controlled by retinoic acid (RA) essential for lung formation. Our genome-wide screen and functional analyses have shown that RA controls lung formation by balancing the effect of Wnt, Tgfb and Fgf10 in induction of lung buds (Chen et al. J. Clin. Invest. 2010). Disruption of these interactions resulted in the lung abnormalities classically reported in vitamin A deficiency. Notably our analyses revealed an unsuspected ectopic formation of airway smooth muscle (SM) in RA-deficient embryonic lungs. Further genetic, organ culture and maternal Vitamin-A dietary restriction approaches showed that RA is required to restrict the SM program in mesenchymal progenitors during airway formation to prevent aberrant differentiation. Moreover, adult mice who had been only briefly exposed to Vitamin A deficiency in uterus had excessive airway SM and developed asthma-like hyperresponsiveness (Chen et al. J Clin Invest 2014; Marquez & Cardoso Mol Cell Ped 2016). Current research focuses on the epigenetic mechanisms involved in these alterations.
Understanding formation and diversification of multiciliated cells
Multiciliated cells are major components of the airway epithelium, contributing through mucociliary clearance to the lung defense mechanisms against environmental agents. Exposure to smoke or biological pathogens can disturb the integrity and function of multiciliated cells. Changes in number, morphology and function of multiciliated cells have been reported in human conditions, including ciliopathies and chronic obstructive pulmonary disease. Multiciliated cells are surprisingly long-lived, lasting for nearly half of the life span of an adult mouse and do not proliferate. Thus, they need to be replaced from a population of local stem/progenitor cells. How multiciliated cells form from respiratory progenitors is still little understood. We have shown that the abundance of multiciliated cells is greatly dependent on Notch (Tsao et al. Development 2009, 2011), and that multiciliogenesis is initiated by the coordination of transcription and cytoplasmic events mediated by the tumor-suppressor E2F4 (Mori et al., Nature Comm 2017; Hazan et al. Mol. Bio.Cell 2021). We are studying the regulation of these events under normal and pathological conditions and the role of other early signals in commitment of airway progenitors to the multiciliated cell phenotype.