Search

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

SummaryLung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNAin situhybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primerTMPRSS2was highest in ciliated cells and type I alveolar epithelial cells (AT1), andTMPRSS2expression was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressingTMPRSS2.Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in the lung epithelium, and suggest that developmental regulation ofTMPRSS2may underlie the relative protection of infants and children from severe respiratory illness.

A pilot program was initiated using whole genome sequencing (WGS) to diagnose suspected genetic disorders in the Genetics Clinic at Children's Hospital of Wisconsin. Twenty-two patients underwent WGS between 2010 and 2013. Initially, we obtained a 14% (3/22) diagnosis rate over 2 years; with subsequent reanalysis, this increased to 36% (8/22). Disease causing variants were identified in SKIV2L, CECR1, DGKE, PYCR2, RYR1, PDGFRB, EFTUD2, and BCS1L. In 75% (6/8) of diagnosed cases, the diagnosis affected treatment and/or medical surveillance. Additionally, one case demonstrated a homozygous A18V variant in VLDLR that appears to be associated with a previously undescribed phenotype.

Although rhinoviral infections, a major cause of asthma exacerbations, occur predominantly in upper airway bronchial epithelial cells, monocytic-lineage cells are implicated in establishing the inflammatory microenvironment observed during the disease. Human rhinovirus (HRV) is unique in that nearly genetically identical viruses bind either the ICAM-1 or low-density lipoprotein receptor (LDL-R). Within minutes of binding, HRV is capable of eliciting a signaling response in both epithelial cells and monocyte-derived macrophages. It is unclear whether this signaling response is important to the subsequent release of inflammatory mediators, particularly in cells not capable of supporting viral replication. We show here that the small molecular mass G-protein Rac is activated following exposure of macrophages to HRV serotypes known to be ICAM-1- and LDL-R-tropic. We demonstrate that inhibiting Rac resulted in the upregulation of TLR3 in macrophages exposed to major- and minor-group HRV, and resulted in increased release of IFN-α. Furthermore, inhibiting Rac in HRV-exposed macrophages attenuated activation of the stress kinase p38 and release of the pro-inflammatory cytokine CCL2, but inhibiting Rac did not affect release of the pro-inflammatory cytokine CCL5. These findings suggest that Rac is an important regulator in establishing the inflammatory microenvironment that is initiated in the human airway upon exposure to rhinovirus.

Establishing a causal link between genomic variation and pathology is the foundation of the successful application of genomics to clinical medicine. Whole genome sequencing (WGS) increases the reso...

Major- and minor-group rhinoviruses enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although ep...

Major- and minor-group human rhinoviruses (HRV) enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group) and HRV1A (minor-group), upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF-2, CREB, CEBP-alpha) were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus.

Whole exome sequencing (WES) is currently used to identify the genetic causes of many diseases, especially monogenic disorders. Ng et al,1 in 2009, completed the first proof-of-principle study demonstrating the feasibility of using exome sequencing to identify causal variants for diseases, specifically Freeman-Sheldon syndrome. Within 2 years, there was a marked increase in publications presenting WES data, and the pace continues to accelerate (Figure). In 2010, WES began to be used for clinical diagnoses, particularly for mendelian disorders. In early 2011, Worthey et al2 used exome sequencing to facilitate clinical diagnosis and modify treatment in a single case. Despite many of the successes resulting from exome sequencing, more than half of the approximately 7000 known or suspected mendelian disorders have not yet been discovered,3 which highlights the need for more genetic, mechanistic, and clinical studies, particularly if the data are to be used clinically. Moreover, as our knowledge of the genome increases, examples of some of the complexities associated with genotypic-phenotypic relationships further substantiate the need for both additional genomic annotation and many more sequenced genomes with phenotypic information. Some of these complexities include the following: (1) Variants in the same genes may lead to different clinical manifestations or phenotypes; (2) what appear to be similar phenotypic observations may result from different causal disease variants operating through distinct pathophysiological mechanisms; and (3) the recent ENCODE (Encyclopedia of DNA Elements) papers, which suggest that up to 80% of the human genome is functional.4 Figure. Number of exome sequencing publications by year. “Human exome sequencing” and “human patient exome sequencing” were searched in PubMed. Reviews, perspectives, and methodology papers were excluded. Article see p 1009 In this issue of Circulation , Crotti and colleagues5 use a …