Identification of lung innervating sensory neurons and their target specificity.

Known as the gas exchange organ, the lung is also critical for responding to the aerosol environment in part through interaction with the nervous system. The diversity and specificity of lung innervating neurons remain poorly understood. Here, we interrogated the cell body location and molecular signature and projection pattern of lung innervating sensory neurons. Retrograde tracing from the lung coupled with whole tissue clearing highlighted neurons primarily in the vagal ganglia. Centrally, they project specifically to the nucleus of the solitary tract in the brainstem. Peripherally, they enter the lung alongside branching airways. Labeling of nociceptor Trpv1+ versus peptidergic Tac1+ vagal neurons showed shared and distinct terminal morphology and targeting to airway smooth muscles, vasculature including lymphatics, and alveoli. Notably, a small population of vagal neurons that are Calb1+ preferentially innervate pulmonary neuroendocrine cells, a demonstrated airway sensor population. This atlas of lung innervating neurons serves as a foundation for understanding their function in lung.

Single-cell multiomic profiling of human lungs reveals cell-type-specific and age-dynamic control of SARS-CoV2 host genes.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases.

Smooth Muscle Differentiation Is Essential for Airway Size, Tracheal Cartilage Segmentation, but Dispensable for Epithelial Branching.

Airway smooth muscle is best known for its role as an airway constrictor in diseases such as asthma. However, its function in lung development is debated. A prevalent model, supported by in vitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to branching tips. Here, we test this model in vivo by inactivating Myocardin, which prevented airway smooth muscle differentiation. We found that Myocardin mutants show normal branching, despite the absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

The respiratory lineage initiates from the specification of NKX2-1+ progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2, which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size.

Crouzon syndrome mouse model exhibits cartilage hyperproliferation and defective segmentation in the developing trachea.

Crouzon syndrome is the result of a gain-of-function point mutation in FGFR2. Mimicking the human mutation, a mouse model of Crouzon syndrome (Fgfr2342Y) recapitulates patient deformities, including failed tracheal cartilage segmentation, resulting in a cartilaginous sleeve in the homozygous mutants. We found that the Fgfr2C342Y/C342Y mutants exhibited an increase in chondrocytes prior to segmentation. This increase is due at least in part to over proliferation. Genetic ablation of chondrocytes in the mutant led to restoration of segmentation in the lateral but not central portion of the trachea. These results suggest that in the Fgfr2C342Y/C342Y mutants, increased cartilage cell proliferation precedes and contributes to the disruption of cartilage segmentation in the developing trachea.

Consider the lung as a sensory organ: A tip from pulmonary neuroendocrine cells.

While the lung is commonly known for its gas exchange function, it is exposed to signals in the inhaled air and responds to them by collaborating with other systems including immune cells and the neural circuit. This important aspect of lung physiology led us to consider the lung as a sensory organ. Among different cell types within the lung that mediate this role, several recent studies have renewed attention on pulmonary neuroendocrine cells (PNECs). PNECs are a rare, innervated airway epithelial cell type that accounts for <1% of the lung epithelium population. They are enriched at airway branch points. Classical in vitro studies have shown that PNECs can respond to an array of aerosol stimuli such as hypoxia, hypercapnia and nicotine. Recent in vivo evidence suggests an essential role of PNECs at neuroimmunomodulatory sites of action, releasing neuropeptides, neurotransmitters and facilitating asthmatic responses to allergen. In addition, evidence supports that PNECs can function both as progenitor cells and progenitor niches following airway epithelial injury. Increases in PNECs have been documented in a large array of chronic lung diseases. They are also the cells-of-origin for small cell lung cancer. A better understanding of the specificity of their responses to distinct insults, their impact on normal lung function and their roles in the pathogenesis of pulmonary ailments will be the next challenge toward designing therapeutics targeting the neuroendocrine system in lung.

Lats1/2 inactivation reveals Hippo function in alveolar type I cell differentiation during lung transition to air breathing.

Lung growth to its optimal size at birth is driven by reiterative airway branching followed by differentiation and expansion of alveolar cell types. How this elaborate growth is coordinated with the constraint of the chest is poorly understood. Here, we investigate the role of Hippo signaling, a cardinal pathway in organ size control, in mouse lung development. Unexpectedly, we found that epithelial loss of the Hippo kinase genes Lats1 and Lats2 (Lats1/2) leads to a striking reduction of lung size owing to an early arrest of branching morphogenesis. This growth defect is accompanied by abnormalities in epithelial cell polarity, cell division plane and extracellular matrix deposition, as well as precocious and increased expression of markers for type 1 alveolar epithelial cells (AEC1s), an indicator of terminal differentiation. Increased AEC1s were also observed in transgenic mice with overexpression of a constitutive nuclear form of downstream transcriptional effector YAP. Conversely, loss of Yap and Taz led to decreased AEC1s, demonstrating that the canonical Hippo signaling pathway is both sufficient and necessary to drive AEC1 fate. These findings together reveal unique roles of Hippo-LATS-YAP signaling in the developing mouse lung.

ß-Catenin signaling is essential for mammalian larynx recanalization and the establishment of vocal fold progenitor cells.

Congenital laryngeal webs result from failure of vocal fold separation during development in utero Infants present with life-threatening respiratory problems at birth, and extensive lifelong difficulties in breathing and voicing. The molecular mechanisms that instruct vocal fold formation are rarely studied. Here, we show, for the first time, that conditional inactivation of the gene encoding ß-catenin in the primitive laryngopharyngeal epithelium leads to failure in separation of the vocal folds, which approximates the gross phenotype of laryngeal webbing. These defects can be traced to a series of morphogenesis defects, including delayed fusion of the epithelial lamina and formation of the laryngeal cecum, failed separation of the larynx and esophagus with reduced and disorganized cartilages and muscles. Parallel to these morphogenesis defects, inactivation of ß-catenin disrupts stratification of epithelial cells and establishment of p63+ basal progenitors. These findings provide the first line of evidence that links ß-catenin function to the cell proliferation and progenitor establishment during larynx and vocal fold development.

Embryology meets molecular biology: Deciphering the apical ectodermal ridge.

More than sixty years ago, while studying feather tracks on the shoulder of the chick embryo, Dr. John Saunders used Nile Blue dye to stain the tissue. There, he noticed a darkly stained line of cells that neatly rims the tip of the growing limb bud. Rather than ignoring this observation, he followed it up by removing this tissue and found that it led to a striking truncation of the limb skeletons. This landmark experiment marks the serendipitous discovery of the apical ectodermal ridge (AER), the quintessential embryonic structure that drives the outgrowth of the limb. Dr. Saunders continued to lead the limb field for the next fifty years, not just through his own work, but also by inspiring the next generation of researchers through his infectious love of science. Together, he and those who followed ushered in the discovery of fibroblast growth factor (FGF) as the AER molecule. The seamless marriage of embryology and molecular biology that led to the decoding of the AER serves as a shining example of how discoveries are made for the rest of the developmental biology field.

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

Syndactyly in a novel Fras1(rdf) mutant results from interruption of signals for interdigital apoptosis.

BACKGROUND: Fras1 encodes an extracellular matrix protein that is critical for the establishment of the epidermal basement membrane during gestation. In humans, mutations in FRAS1 cause Fraser Syndrome (FS), a pleiotropic condition with many clinical presentations such as limb, eye, kidney, and craniofacial deformations. Many of these defects are mimicked by loss of Fras1 in mice, and are preceded by the formation of epidermal blisters in utero. RESULTS: In this study, we identified a novel ENU-derived rounded foot (rdf) mouse mutant with highly penetrant hindlimb soft-tissue syndactyly, among other structural defects. Mapping and sequencing revealed that rdf is a novel loss-of-function nonsense allele of Fras1 (Fras1(rdf)). Focusing on the limb, we found that the Fras1(rdf) syndactyly phenotype originates from loss of interdigital cell death (ICD). Despite normal expression of bone morphogenetic protein (BMP) ligands and their receptors, the BMP downstream target gene Msx2, which is also necessary and sufficient to promote ICD, was down-regulated in the interdigital regions of Fras1(rdf) hindlimb buds. CONCLUSIONS: The close correlation between limb bud epidermal blistering, decreased Msx2 expression, and reduced ICD in the Fras1(rdf) hindlimb buds suggests that epithelium detachment from the mesenchyme may create a physical gap that interrupts the transmission of BMP, among other signals, resulting in soft tissue syndactyly.

Pulmonary neuroendocrine cells function as airway sensors to control lung immune response.

The lung is constantly exposed to environmental atmospheric cues. How it senses and responds to these cues is poorly defined. Here, we show that Roundabout receptor (Robo) genes are expressed in pulmonary neuroendocrine cells (PNECs), a rare, innervated epithelial population. Robo inactivation in mouse lung results in an inability of PNECs to cluster into sensory organoids and triggers increased neuropeptide production upon exposure to air. Excess neuropeptides lead to an increase in immune infiltrates, which in turn remodel the matrix and irreversibly simplify the alveoli. We demonstrate in vivo that PNECs act as precise airway sensors that elicit immune responses via neuropeptides. These findings suggest that the PNEC and neuropeptide abnormalities documented in a wide array of pulmonary diseases may profoundly affect symptoms and progression.

Genomic Analyses of Sperm Fate Regulator Targets Reveal a Common Set of Oogenic mRNAs in Caenorhabditis elegans.

Germ cell specification as sperm or oocyte is an ancient cell fate decision, but its molecular regulation is poorly understood. In Caenorhabditis elegans, the FOG-1 and FOG-3 proteins behave genetically as terminal regulators of sperm fate specification. Both are homologous to well-established RNA regulators, suggesting that FOG-1 and FOG-3 specify the sperm fate post-transcriptionally. We predicted that FOG-1 and FOG-3, as terminal regulators of the sperm fate, might regulate a battery of gamete-specific differentiation genes. Here we test that prediction by exploring on a genomic scale the messenger RNAs (mRNAs) associated with FOG-1 and FOG-3. Immunoprecipitation of the proteins and their associated mRNAs from spermatogenic germlines identifies 81 FOG-1 and 722 FOG-3 putative targets. Importantly, almost all FOG-1 targets are also FOG-3 targets, and these common targets are strongly biased for oogenic mRNAs. The discovery of common target mRNAs suggested that FOG-1 and FOG-3 work together. Consistent with that idea, we find that FOG-1 and FOG-3 proteins co-immunoprecipitate from both intact nematodes and mammalian tissue culture cells and that they colocalize in germ cells. Taking our results together, we propose a model in which FOG-1 and FOG-3 work in a complex to repress oogenic transcripts and thereby promote the sperm fate.

A three-dimensional study of alveologenesis in mouse lung.

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simplification in BPD.

FGF-Regulated ETV Transcription Factors Control FGF-SHH Feedback Loop in Lung Branching.

The mammalian lung forms its elaborate tree-like structure following a largely stereotypical branching sequence. While a number of genes have been identified to play essential roles in lung branching, what coordinates the choice between branch growth and new branch formation has not been elucidated. Here we show that loss of FGF-activated transcription factor genes, Etv4 and Etv5 (collectively Etv), led to prolonged branch tip growth and delayed new branch formation. Unexpectedly, this phenotype is more similar to mutants with increased rather than decreased FGF activity. Indeed, an increased Fgf10 expression is observed, and reducing Fgf10 dosage can attenuate the Etv mutant phenotype. Further evidence indicates that ETV inhibits Fgf10 via directly promoting Shh expression. SHH in turn inhibits local Fgf10 expression and redirects growth, thereby initiating new branches. Together, our findings establish ETV as a key node in the FGF-ETV-SHH inhibitory feedback loop that dictates branching periodicity.

Ontogeny of the mouse vocal fold epithelium.

This investigation provides the first systematic determination of the cellular and molecular progression of vocal fold (VF) epithelium development in a murine model. We define five principal developmental events that constitute the progression from VF initiation in the embryonic anterior foregut tube to fully differentiated and functional adult tissue. These developmental events include (1) the initiation of the larynx and vocal folds with apposition of the lateral walls of the primitive laryngopharynx (embryonic (E) day 10.5); (2) the establishment of the epithelial lamina with fusion of the lateral walls of the primitive laryngopharynx (E11.5); (3) the epithelial lamina recanalization and separation of VFs (E13.5-18.5); (4) the stratification of the vocal folds (E13.5-18.5); and (5) the maturation of vocal fold epithelium (postnatal stages). The illustration of these morphogenetic events is substantiated by dynamic changes in cell proliferation and apoptosis, as well as the expression pattern of key transcription factors, FOXA2, SOX2 and NKX2-1 that specify and pattern the foregut endoderm. Furthermore, we documented the gradual conversion of VF epithelial cells from simple precursors expressing cytokeratins 8 and 18 in the embryo into mature stratified epithelial cells also expressing cytokeratins 5 and 14 in the adult. Interestingly, in the adult, cytokeratins 5 and 14 appear to be expressed in all cell layers in the VF, in contrast to their preferential localization to the basal cell layer in surrounding epithelium. To begin investigating the role of signaling molecules in vocal fold development, we characterized the expression pattern of SHH pathway genes, and how loss of Shh affects vocal fold development in the mutant. This study defines the cellular and molecular context and serves as the necessary foundation for future functional investigations of VF formation.

Comparison of temporal transcriptomic profiles from immature lungs of two rat strains reveals a viral response signature associated with chronic lung dysfunction.

Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs.

Alterations in microRNA-124 and AMPA receptors contribute to social behavioral deficits in frontotemporal dementia.

Neurodegenerative diseases, such as frontotemporal dementia (FTD), are often associated with behavioral deficits, but the underlying anatomical and molecular causes remain poorly understood. Here we show that forebrain-specific expression of FTD-associated mutant CHMP2B in mice causes several age-dependent neurodegenerative phenotypes, including social behavioral impairments. The social deficits were accompanied by a change in AMPA receptor (AMPAR) composition, leading to an imbalance between Ca(2+)-permeable and Ca(2+)-impermeable AMPARs. Expression of most AMPAR subunits was regulated by the brain-enriched microRNA miR-124, whose abundance was markedly decreased in the superficial layers of the cerebral cortex of mice expressing the mutant CHMP2B. We found similar changes in miR-124 and AMPAR levels in the frontal cortex and induced pluripotent stem cell-derived neurons from subjects with behavioral variant FTD. Moreover, ectopic miR-124 expression in the medial prefrontal cortex of mutant mice decreased AMPAR levels and partially rescued behavioral deficits. Knockdown of the AMPAR subunit Gria2 also alleviated social impairments. Our results identify a previously undescribed mechanism involving miR-124 and AMPARs in regulating social behavior in FTD and suggest a potential therapeutic avenue.

Establishment of smooth muscle and cartilage juxtaposition in the developing mouse upper airways.

In the trachea and bronchi of the mouse, airway smooth muscle (SM) and cartilage are localized to complementary domains surrounding the airway epithelium. Proper juxtaposition of these tissues ensures a balance of elasticity and rigidity that is critical for effective air passage. It is unknown how this tissue complementation is established during development. Here we dissect the developmental relationship between these tissues by genetically disrupting SM formation (through Srf inactivation) or cartilage formation (through Sox9 inactivation) and assessing the impact on the remaining lineage. We found that, in the trachea and main bronchi, loss of SM or cartilage resulted in an increase in cell number of the remaining lineage, namely the cartilage or SM, respectively. However, only in the main bronchi, but not in the trachea, did the loss of SM or cartilage lead to a circumferential expansion of the remaining cartilage or SM domain, respectively. In addition to SM defects, cartilage-deficient tracheas displayed epithelial phenotypes, including decreased basal cell number, precocious club cell differentiation, and increased secretoglobin expression. These findings together delineate the mechanisms through which a cell-autonomous disruption of one structural tissue can have widespread consequences on upper airway function.