Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype
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
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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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 2

Single-cell analysis of GLI2+ mesenchyme in the adult lung.

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Single-cell analysis of GLI2+ mesenchyme in the adult lung. (A) Unbiased...
(A) Unbiased clustering of transcriptomes of individual GLI2+ cells from the adult lung. Each cell is represented as a single dot that is colored by the clustering algorithm and plotted on the tSNE graph. Four distinct clusters emerge from a graph-based clustering algorithm with the majority of the cells in cluster 1 (C1) and cluster 2 (C2). (B) The top expressed genes in cluster 1 are enriched for the GO term extracellular matrix, while the cluster 2 gene signature is enriched for the GO term cell-to-cell signaling. Size of the dot plot represents proportion of cells within the cluster expressing the gene, and color denotes the level of expression (Exp.). Prox, proximal; Dist, distal. (C) Flow cytometry analysis of the Gli1:Gli2 reporter (Gli1EGFP:Gli2creERT2-tdT/+) shows that the cluster 1 markers LY6A and LY6C1 detect cells that are predominantly in the Hhhi (GFP+, GLI1+GLI2+) fraction of the GLI2+ mesenchyme, whereas the cluster 2 marker ITGA8 detects cells predominantly in the Hhlo (GFP–, GLI1–GLI2+) fraction. (D) Schematic of the experimental flow to isolate GLI2+ cells where the Hh-activation domain is expanded to the distal alveoli for bulk RNA-seq, with n = 4 per group. Tam, tamoxifen. (E) The mean difference plot displays the log-fold differences (y axis) versus the mean counts for all genes in the bulk RNA-seq experiment, with each dot representing a gene detected in the GLI2+ mesenchyme. The majority of the proximal genes are upregulated in the Hh-expanded Gli2+ mesenchyme, while the majority of the distal genes are downregulated. Results were replicated (n ≥ 2 experiments).