Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype
Chaoqun Wang, … , Harold A. Chapman, Tien Peng
Chaoqun Wang, … , Harold A. Chapman, Tien Peng
Published July 12, 2018
Citation Information: J Clin Invest. 2018;128(10):4343-4358. https://doi.org/10.1172/JCI99435.
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Research Article Development Pulmonology

Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype

Abstract

GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.

Authors

Chaoqun Wang, Nabora S. Reyes de Mochel, Stephanie A. Christenson, Monica Cassandras, Rebecca Moon, Alexis N. Brumwell, Lauren E. Byrnes, Alfred Li, Yasuyuki Yokosaki, Peiying Shan, Julie B. Sneddon, David Jablons, Patty J. Lee, Michael A. Matthay, Harold A. Chapman, Tien Peng

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Figure 1

Proximal-distal asymmetry of Hh activation in the adult lung mesenchyme.

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Proximal-distal asymmetry of Hh activation in the adult lung mesenchyme....
(A) Gli1LacZ/+ reporter demonstrates Gli1 expression largely confined to the proximal airway, whereas Gli2LacZ/+ reporter demonstrates Gli2 expression both in the proximal airway and the distal alveoli. (B) Generation of double reporters (Gli1LacZ/+:PdgfraGFP/+ and Gli2LacZ/+:PdgfraGFP/+) demonstrates that GLI1+ cells constitute a significant portion of the PDGFRα+ proximal airway mesenchymal fibroblasts, but do not contribute to the distal mesenchymal fibroblast population. In contrast, GLI2+ cells contribute to both the proximal and distal mesenchyme (arrows, overlap expression). (C) Generation of Gli2creERT2-tdT/+:R26RYFP/+:Ptch1LacZ/+ followed by lineage labeling of GLI2+ cells shows that Gli2 and Ptch1 expression largely overlaps in both the proximal and distal mesenchyme. (D) Left and center: utilizing fluorescent reporters of Gli1 and Gli2 (Gli1EGFP and Gli2creERT2-tdT/+), we confirmed that GLI1+ and GLI2+ cells contribute to a large proportion of the PDGFRα+ mesenchyme in the lung. Right: crossing the 2 reporters generates a dual color Gli1:Gli2 reporter (Gli1EGFP:Gli2creERT2-tdT/+), which shows that almost all of the GLI1+ cells coexpress GLI2 and that the GLI2 domain can be divided into proximal GLI1+ and distal GLI1– subpopulations. (E) qPCR analysis demonstrates significant enrichment of Hh target genes Gli1, Ptch1, and Ptch2 in the sorted proximal mesenchyme (GLI1+GLI2+) relative to distal (GLI1–GLI2+). Statistical analysis was done using 1-tailed paired Student’s t test. *P < 0.05. (F) Schematic representing a broad GLI2+ mesenchyme in the adult lung segregated by asymmetric activation of Hh along the proximal-distal axis. Scale bars: 100 μm. Data are represented as mean ± SEM, with n = 3 per group. Results were replicated (n ≥ 2 experiments).