Michael P. Morley

Long noncoding RNAs are spatially correlated with transcription factors and regulate lung development

Long noncoding RNAs (lncRNAs) are thought to play important roles in regulating gene transcription, but few have well-defined expression patterns or known biological functions during mammalian development. Using a conservative pipeline to identify lncRNAs that have important biological functions, we identified 363 lncRNAs in the lung and foregut endoderm. Importantly, we show that these lncRNAs are spatially correlated with transcription factors across the genome. In-depth expression analyses of lncRNAs with genomic loci adjacent to the critical transcription factors Nkx2.1, Gata6, Foxa2 (forkhead box a2), and Foxf1 mimic the expression patterns of their protein-coding neighbor. Loss-of-function analysis demonstrates that two lncRNAs, LL18/NANCI (Nkx2.1-associated noncoding intergenic RNA) and LL34, play distinct roles in endoderm development by controlling expression of critical developmental transcription factors and pathways, including retinoic acid signaling. In particular, we show that LL18/NANCI acts upstream of Nkx2.1 and downstream from Wnt signaling to regulate lung endoderm gene expression. These studies reveal that lncRNAs play an important role in foregut and lung endoderm development by regulating multiple aspects of gene transcription, often through regulation of transcription factor expression.

Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration

Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Although previous studies have demonstrated that certain embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover. Here we demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signalling. Epithelial-specific deletion of sonic hedgehog (Shh) during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signalling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signalling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial–mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

Development and Regeneration of Sox2+ Endoderm Progenitors Are Regulated by a HDAC1/2-Bmp4/Rb1 Regulatory Pathway

The mechanisms that govern the maintenance and differentiation of tissue-specific progenitors in development and tissue regeneration are poorly understood. We show that development of Sox2+ progenitors in the lung endoderm is regulated by histone deacetylases 1 and 2 (Hdac1/2). Hdac1/2 deficiency leads to a loss of Sox2 expression and a block in proximal airway development. This is mediated in part by derepression of Bmp4 and the tumor suppressor Rb1, which are direct transcriptional targets of Hdac1/2. In contrast to development, postnatal loss of Hdac1/2 in airway epithelium does not affect the expression of Sox2 or Bmp4. However, postnatal loss of Hdac1/2 leads to increased expression of the cell-cycle regulators Rb1, p21/Cdkn1a, and p16/Ink4a, resulting in a loss of cell-cycle progression and defective regeneration of Sox2+ lung epithelium. Thus, Hdac1/2 have both common and unique targets that differentially regulate tissue-specific progenitor activity during development and regeneration.

Tissue-Specific RNA-Seq in Human Evoked Inflammation Identifies Blood and Adipose LincRNA Signatures of Cardiometabolic Diseases

Objective—Inappropriate transcriptional activation of innate immunity is a pathological feature of several cardiometabolic disorders, but little is known about inflammatory modulation of long intergenic noncoding RNAs (lincRNAs) in disease-relevant human tissues. Approach and Results—We applied deep RNA sequencing (>500 million filtered reads per sample) to blood and adipose during low-dose experimental endotoxemia (lipopolysaccharide) in a healthy human, with targeted replication in separate individuals undergoing endotoxemia (n=6), to identify inflammatory lincRNAs. A subset of these lincRNAs was examined for expression in adipocytes and monocytes, modulation in adipose of obese humans, and overlap with genome-wide association study signals for inflammatory and cardiometabolic traits. Of a stringent set of 4284 lincRNAs, ≈11% to 22% were expressed with 201 and 56 lincRNAs modulated by lipopolysaccharide in blood or adipose, respectively. Tissue-specific expression of a subset of 6 lipopolysaccharide-lincRNAs was replicated with lipopolysaccharide modulation confirmed for all 3 expressed in blood and 2 of 4 expressed in adipose. The broader generalizability of findings in blood of subject A was confirmed by RNA sequencing in 7 additional subjects. We confirmed adipocytes and monocytes as potential cell-sources of selective lipopolysaccharide-regulated lincRNAs, and 2 of these, linc-DMRT2 (P=0.002) and linc-TP53I13 (P=0.01), were suppressed in adipose of obese humans. Finally, we provide examples of lipopolysaccharide-modulated lincRNAs that overlap single nucleotide polymorphisms that are associated with cardiometabolic traits. Conclusions—Our findings provide novel insights into tissue-level, inflammatory transcriptome regulation in cardiometabolic diseases. These are complementary to more usual approaches limited to interrogation of DNA variations.

Distinct mesenchymal lineages and niches promote epithelial self-renewal and myofibrogenesis in the lung

The lung is an architecturally complex organ comprising a heterogeneous mixture of various epithelial and mesenchymal lineages. We use single-cell RNA sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The mesenchymal alveolar niche cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ myofibrogenic progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells, including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways in promoting self-renewal versus a pathological response to tissue injury.

Gene expression and genetic variation in human atria

BACKGROUND The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood. OBJECTIVE The purpose of this study was to characterize gene expression and genetic variation in human atria. METHODS We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0. RESULTS We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a single nucleotide polymorphism at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. CONCLUSION We found a distinct transcriptional profile between the right and left atrium and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Ezh2 represses the basal cell lineage during lung endoderm development

The development of the lung epithelium is regulated in a stepwise fashion to generate numerous differentiated and stem cell lineages in the adult lung. How these different lineages are generated in a spatially and temporally restricted fashion remains poorly understood, although epigenetic regulation probably plays an important role. We show that the Polycomb repressive complex 2 component Ezh2 is highly expressed in early lung development but is gradually downregulated by late gestation. Deletion of Ezh2 in early lung endoderm progenitors leads to the ectopic and premature appearance of Trp63+ basal cells that extend the entire length of the airway. Loss of Ezh2 also leads to reduced secretory cell differentiation. In their place, morphologically similar cells develop that express a subset of basal cell genes, including keratin 5, but no longer express high levels of either Trp63 or of standard secretory cell markers. This suggests that Ezh2 regulates the phenotypic switch between basal cells and secretory cells. Together, these findings show that Ezh2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation. Highlighted article: The polycomb component Ezh2 regulates the temporal and spatial patterning of the lung, acting to control the phenotypic switch between basal and secretory cells.

Wnt ligand/Frizzled 2 receptor signaling regulates tube shape and branch-point formation in the lung through control of epithelial cell shape

Significance We generated a conditional mouse allele for the Wnt receptor Fzd2 and used it to assess the role of Fzd2-mediated Wnt signaling in the lung. Loss of Fzd2 specifically in the developing lung epithelium results in defects in domain branch-point formation which alter the primary branching program of the lung. We show that Fzd2 is required to sculpt the developing epithelium in the lung through activation of the small GTPase RhoA and control of epithelial cell shape. These results reveal the importance of Wnt/RhoA signaling in altering the shape of the developing epithelium of branched organs such as the lung. Such studies highlight the interconnectedness of signaling pathways during the formation of a branched network. Changing the morphology of a simple epithelial tube to form a highly ramified branching network requires changes in cell behavior that lead to tissue-wide changes in organ shape. How epithelial cells in branched organs modulate their shape and behavior to promote bending and sculpting of the epithelial sheet is not well understood, and the mechanisms underlying this process remain obscure. We show that the Wnt receptor Frizzled 2 (Fzd2) is required for domain branch formation during the initial establishment of the respiratory tree. Live imaging and transcriptome analysis of lung-branching morphogenesis demonstrate that Fzd2 promotes changes in epithelial cell length and shape. These changes in cell morphology deform the developing epithelial tube to generate and maintain new domain branches. Fzd2 controls branch formation and the shape of the epithelial tube by regulating Rho signaling and by the localization of phospho-myosin light chain 2, in turn controlling the changes in the shape of epithelial cells during morphogenesis. This study demonstrates the importance of Wnt/Fzd2 signaling in promoting and maintaining changes in epithelial cell shape that affect development of a branching network.

Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor

Functional tissue regeneration is required for the restoration of normal organ homeostasis after severe injury. Some organs, such as the intestine, harbour active stem cells throughout homeostasis and regeneration; more quiescent organs, such as the lung, often contain facultative progenitor cells that are recruited after injury to participate in regeneration. Here we show that a Wnt-responsive alveolar epithelial progenitor (AEP) lineage within the alveolar type 2 cell population acts as a major facultative progenitor cell in the distal lung. AEPs are a stable lineage during alveolar homeostasis but expand rapidly to regenerate a large proportion of the alveolar epithelium after acute lung injury. AEPs exhibit a distinct transcriptome, epigenome and functional phenotype and respond specifically to Wnt and Fgf signalling. In contrast to other proposed lung progenitor cells, human AEPs can be directly isolated by expression of the conserved cell surface marker TM4SF1, and act as functional human alveolar epithelial progenitor cells in 3D organoids. Our results identify the AEP lineage as an evolutionarily conserved alveolar progenitor that represents a new target for human lung regeneration strategies.

HDAC3-Dependent Epigenetic Pathway Controls Lung Alveolar Epithelial Cell Remodeling and Spreading via miR-17-92 and TGF-β Signaling Regulation

The terminal stages of pulmonary development, called sacculation and alveologenesis, involve both differentiation of distal lung endoderm progenitors and extensive cellular remodeling of the resultant epithelial lineages. These processes are coupled with dramatic expansion of distal airspace and surface area. Despite the importance of these late developmental processes and their relation to neonatal respiratory diseases, little is understood about the molecular and cellular pathways critical for their successful completion. We show that a histone deacetylase 3 (Hdac3)-mediated epigenetic pathway is critical for the proper remodeling and expansion of the distal lung saccules into primitive alveoli. Loss of Hdac3 in the developing lung epithelium leads to a reduction of alveolar type 1 cell spreading and a disruption of lung sacculation. Hdac3 represses miR-17-92 expression, a microRNA cluster that regulates transforming growth factor β (TGF-β) signaling. De-repression of miR-17-92 in Hdac3-deficient lung epithelium results in decreased TGF-β signaling activity. Importantly, inhibition of TGF-β signaling and overexpression of miR-17-92 can phenocopy the defects observed in Hdac3 null lungs. Conversely, loss of miR-17-92 expression rescues many of the defects caused by loss of Hdac3 in the lung. These studies reveal an intricate epigenetic pathway where Hdac3 is required to repress miR-17-92 expression to allow for proper TGF-β signaling during lung sacculation.