The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

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.

Extensive Recombination Suppression and Epistatic Selection Causes Chromosome-Wide Differentiation of a Selfish Sex Chromosome in Drosophila pseudoobscura.

Sex-Ratio (SR) chromosomes are selfish X-chromosomes that distort Mendelian segregation and are commonly associated with inversions. These chromosomal rearrangements suppress recombination with Standard (ST) X-chromosomes and are hypothesized to maintain multiple alleles important for distortion in a single large haplotype. Here, we conduct a multifaceted study of the multiply inverted Drosophila pseudoobscura SR chromosome to understand the evolutionary history, genetic architecture, and present-day dynamics that shape this enigmatic selfish chromosome. The D. pseudoobscura SR chromosome has three nonoverlapping inversions of the right arm of the metacentric X-chromosome: basal, medial, and terminal. We find that 23 of 29 Mb of the D. pseudoobscuraX-chromosome right arm is highly differentiated between the Standard and SR arrangements, including a 6.6 Mb collinear region between the medial and terminal inversions. Although crossing-over is heavily suppressed on this chromosome arm, we discover it is not completely eliminated, with measured rates indicating recombination suppression alone cannot explain patterns of differentiation or the near-perfect association of the three SR chromosome inversions in nature. We then demonstrate the ancient basal and medial inversions of the SR chromosome contain genes sufficient to cause weak distortion. In contrast, the younger terminal inversion cannot distort by itself, but contains at least one modifier gene necessary for full manifestation of strong sex chromosome distortion. By parameterizing population genetic models for chromosome-wide linkage disequilibrium with our experimental results, we infer that strong selection acts to maintain the near-perfect association of SR chromosome inversions in present-day populations. Based on comparative genomic analyses, direct recombination experiments, segregation distortion assays, and population genetic modeling, we conclude the combined action of suppressed recombination and strong, ongoing, epistatic selection shape the D. pseudoobscura SR arrangement into a highly differentiated chromosome.

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.

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.

Ancestral polymorphisms explain the role of chromosomal inversions in speciation.

Understanding the role of chromosomal inversions in speciation is a fundamental problem in evolutionary genetics. Here, we perform a comprehensive reconstruction of the evolutionary histories of the chromosomal inversions in Drosophila persimilis and D. pseudoobscura. We provide a solution to the puzzling origins of the selfish Sex-Ratio arrangement in D. persimilis and uncover surprising patterns of phylogenetic discordance on this chromosome. These patterns show that, contrary to widely held views, all fixed chromosomal inversions between D. persimilis and D. pseudoobscura were already present in their ancestral population long before the species split. Our results suggest that patterns of higher genomic divergence and an association of reproductive isolation genes with chromosomal inversions may be a direct consequence of incomplete lineage sorting of ancestral polymorphisms. These findings force a reconsideration of the role of chromosomal inversions in speciation, not as protectors of existing hybrid incompatibilities, but as fertile grounds for their formation.

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.

Can We Identify Genes with Increased Phylogenetic Reliability?

Topological heterogeneity among gene trees is widely observed in phylogenomic analyses and some of this variation is likely caused by systematic error in gene tree estimation. Systematic error can be mitigated by improving models of sequence evolution to account for all evolutionary processes relevant to each gene or identifying those genes whose evolution best conforms to existing models. However, the best method for identifying such genes is not well established. Here, we ask if filtering genes according to their clock-likeness or posterior predictive effect size (PPES, an inference-based measure of model violation) improves phylogenetic reliability and congruence. We compared these approaches to each other, and to the common practice of filtering based on rate of evolution, using two different metrics. First, we compared gene-tree topologies to accepted reference topologies. Second, we examined topological similarity among gene trees in filtered sets. Our results suggest that filtering genes based on clock-likeness and PPES can yield a collection of genes with more reliable phylogenetic signal. For the two exemplar data sets we explored, from yeast and amniotes, clock-likeness and PPES outperformed rate-based filtering in both congruence and reliability.