Felicia Chen

Inhibition of Tgfβ signaling by endogenous retinoic acid is essential for primary lung bud induction

Disruption of retinoic acid (RA) signaling during early development results in severe respiratory tract abnormalities, including lung agenesis. Previous studies suggest that this might result from failure to selectively induce fibroblast growth factor 10 (Fgf10) in the prospective lung region of the foregut. Little is known about the RA-dependent pathways present in the foregut that may be crucial for lung formation. By performing global gene expression analysis of RA-deficient foreguts from a genetic [retinaldehyde dehydrogenase 2 (Raldh2)-null] and a pharmacological (BMS493-treated) mouse model, we found upregulation of a large number of Tgfβ targets. Increased Smad2 phosphorylation further suggested that Tgfβ signaling was hyperactive in these foreguts when lung agenesis was observed. RA rescue of the lung phenotype was associated with low levels of Smad2 phosphorylation and downregulation of Tgfβ targets in Raldh2-null foreguts. Interestingly, the lung defect that resulted from RA-deficiency could be reproduced in RA-sufficient foreguts by hyperactivating Tgfβ signaling with exogenous TGFβ1. Preventing activation of endogenous Tgfβ signaling with a pan-specific TGFβ-blocking antibody allowed bud formation and gene expression in the lung field of both Raldh2-null and BMS493-treated foreguts. Our data support a novel mechanism of RA-Tgfβ-Fgf10 interactions in the developing foregut, in which endogenous RA controls Tgfβ activity in the prospective lung field to allow local expression of Fgf10 and induction of lung buds.

A retinoic acid–dependent network in the foregut controls formation of the mouse lung primordium

The developmental abnormalities associated with disruption of signaling by retinoic acid (RA), the biologically active form of vitamin A, have been known for decades from studies in animal models and humans. These include defects in the respiratory system, such as lung hypoplasia and agenesis. However, the molecular events controlled by RA that lead to formation of the lung primordium from the primitive foregut remain unclear. Here, we present evidence that endogenous RA acts as a major regulatory signal integrating Wnt and Tgfbeta pathways in the control of Fgf10 expression during induction of the mouse primordial lung. We demonstrated that activation of Wnt signaling required for lung formation was dependent on local repression of its antagonist, Dickkopf homolog 1 (Dkk1), by endogenous RA. Moreover, we showed that simultaneously activating Wnt and repressing Tgfbeta allowed induction of both lung buds in RA-deficient foreguts. The data in this study suggest that disruption of Wnt/Tgfbeta/Fgf10 interactions represents the molecular basis for the classically reported failure to form lung buds in vitamin A deficiency.

γ-Secretase Activation of Notch Signaling Regulates the Balance of Proximal and Distal Fates in Progenitor Cells of the Developing Lung

Little is known about the mechanisms by which the lung epithelial progenitors are initially patterned and how proximal-distal boundaries are established and maintained when the lung primordium forms and starts to branch. Here we identified a number of Notch pathway components in respiratory progenitors of the early lung, and we investigated the role of Notch in lung pattern formation. By preventing γ-secretase cleavage of Notch receptors, we have disrupted global Notch signaling in the foregut and in the lung during the initial stages of murine lung morphogenesis. We demonstrate that Notch signaling is not necessary for lung bud initiation; however, Notch is required to maintain a balance of proximal-distal cell fates at these early stages. Disruption of Notch signaling dramatically expands the population of distal progenitors, altering morphogenetic boundaries and preventing formation of proximal structures. Our data suggest a novel mechanism in which Notch and fibroblast growth factor signaling interact to control the proximal-distal pattern of forming airways in the mammalian lung.

Mechanisms of Lung Development Contribution to Adult Lung Disease and Relevance to Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) results in major remodeling of the distal airspaces and changes in the differentiation profile of the airway epithelium. The cellular and molecular mechanisms involved in initiation and progression of this disease are little understood. Although environmental factors, including cigarette smoke, have been directly implicated in the pathogenesis of COPD, genetic risk factors also appear to play a fundamental role in the individuals susceptibility to this disease. Lung development depends on precise coordination of signals, such as fibroblast growth factors (Fgf), Sonic Hedgehog (Shh), retinoic acid, Notch, and Tgf beta. Dramatic changes in the pattern of branching and differentiation of the lung epithelium results from disruption of these signals in genetically altered mice. Recent studies, including whole-genome expression and genome-wide association analyses, suggest that some molecular regulators originally described in developmental processes may be altered in patients with COPD. Whether disturbances in the molecular and cellular events mediated by these genes during development participate in the initiation or exacerbation of COPD, needs further investigation. The role of selected pathways, including Sonic hedgehog, Notch, retinoid, and Tgf beta in the developing lung and the potential association with COPD are discussed.

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

Stem Cells in Lung Injury and Repair.

In this review, we summarize the recent literature on the biology of endogenous stem cells in adult lung injury repair. We focus on inxa0vivo studies in mice with an emphasis on data generated using cell-specific Cre-dependent lineage-tracing systems. These studies provide new information on the identification of lung stem cells, their hierarchical relationships, the plasticity of their behavior in different types of injury, and the molecular signals that control their fates. Although most of this work has been on epithelial hierarchies, we expect that further development of robust genetic tools will foster meaningful investigations into how nonepithelial cell populations are controlled during lung injury repair in adults. The ultimate challenge will be to translate these findings to the pathogenesis and treatment of human lung diseases.

Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

Retinoic acid signaling is essential for airway smooth muscle homeostasis

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-β signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-β activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.

Culture of Mouse Embryonic Foregut Explants

The ability to culture embryonic organ rudiments and follow their development ex vivo has helped to understand how tissues are constructed and what cellular and biological events are important in this process. Here we outline a technique for isolation and ex vivo growth of foregut explants from E8.5 mouse embryos. This technique serves as a reliable tool for the analysis of the morphogenetic processes and signaling networks during early development of foregut derivatives, such as the lungs.