Airway basal cells show regionally distinct potential to undergo metaplastic differentiation
- Columbia Center for Human Development, Columbia University Irving Medical Center, United States
- Department of Medicine, Pulmonary Allergy Critical Care, Columbia University Irving Medical Center, United States
- Department of Genetics and Development, Columbia University Irving Medical Center, United States
- Department of Pharmacy Practice and Science, College of Pharmacy, University of Arizona, United States
- Neonatology and Pulmonary Biology Perinatal Institute, Cincinnati Children’s Hospital Medical Center and University of Cincinnati, College of Medicine, United States
- Departments of Systems Biology, Biochemistry & Molecular Biophysics, Biomedical Informatics, Medicine; JP Sulzberger Columbia Genome Center; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, United States
Figures
Figure 1 with 5 supplements
Single cell RNA sequencing (scRNA-Seq) reveals six distinct airway basal cell (BC) subpopulations in the murine trachea.
(A) Diagram: strategy for labeling, isolation and scRNA-Seq analysis of BCs from Trp63CreERT2; Rosa26lox-STOP-lox-tdTomato adult tracheas. (B) Immunofluorescence (IF) of tracheal sections showing …
Figure 1—figure supplement 1
scRNA-Seq of sorted mouse tracheal BCs reveals six distinct subpopulations.
(A) Gating strategy to sort basal cells (BCs) from Trp63CreERT2; Rosa26lox-STOP-lox-tdTomato adult tracheas for single cell RNA sequencing (scRNA-Seq). (B) IF of cytospin of sorted tdTom airway BCs …
Figure 1—figure supplement 2
Mouse tracheal BC subpopulations exhibit distinct gene expression patterns.
(A) Violin plots of single cell RNA sequencing (scRNA-Seq) clusters for general epithelial marker Epcam, respiratory epithelial lineage Nkx2-1 marker, canonical BC markers Trp63, Krt5, Krt15, …
Figure 1—figure supplement 3
tSNE visualization of airway basal cell (BC) single cell RNA sequencing (scRNA-Seq) for BC markers identified from previous publications in mice and humans, colored by expression [log2(TPM +1)] of each marker gene.
Figure 1—figure supplement 4
Basal cell (BC) subtypes showing unique gene expression signatures.
(A) Top 50 genes significantly enriched in BC-1 and BC-2 showing marked differences in expression between these two clusters. (B) Transcription factors differentially enriched in BC-1 or BC-2 …
Figure 1—figure supplement 5
Basal-squamous cluster showing unique gene expression signature compared to the other five basal cell (BC) clusters.
(A) Volcano plot showing differentially expressed in the Basal-squamous cluster vs the other BCs (squamous-associated marker genes depicted in red dots). (B) Enriched KEGG gene sets significantly …
Figure 2 with 2 supplements
BC-1 and BC-2 reside in distinct niches of the adult tracheal epithelium.
(A) Schematic representation of the differential distribution of BC-1 and BC-2 markers in basal cells along the dorsal-ventral axis of the adult trachea (diagram: sm, smooth muscle; ct, cartilage). …
Figure 2—figure supplement 1
BC-1 and BC-2 populations reside in regionally distinct niches of the adult tracheal epithelium.
Serial cross-sections of adult mouse trachea immunostained for BC-1 and BC-2 markers, KRT5 and a-SMA (identifies smooth muscle layer in dorsal trachea). Differential enrichment of BC-1 and BC-2 …
Figure 2—figure supplement 2
Basal cell (BC) subpopulations other than BC-1 and BC-2 are relatively rare and have no consistent spatial distribution in the adult tracheal epithelium.
Representative tile scan pictures of adult mouse tracheal sections immunostained for KRT5, TRP63 and representative markers of Basal-proliferating (A), Basal-squamous (B), Basal-mesenchyme-like (C), …
Figure 3 with 1 supplement
BC-1 and BC-2 differ in their ability to form organoids and activate a metaplastic program of differentiation in primary cultures.
(A) Strategy for isolation, expansion and analysis of BC-1 and BC-2 in submerged cultures. IF at day 7 showing preserved differential enrichment of BC-1 and BC-2 markers in cultures derived from …
Figure 3—figure supplement 1
Absence of mesenchymal contamination in organoid cultures.
(A) IF of Vimentin (VIM), E-Cadherin (CDH1), and KRT5 in day 14 BC-1 (ventral) or BC-2 (dorsal)-derived organoids showing extensive CDH1 epithelial labeling and no evidence of VIM + cells in these …
Figure 4 with 1 supplement
Differential induction of squamous-associated markers in retinoid-deficient BC-2-derived organotypic cultures.
Expansion and differentiation of BC-1 and BC-2 populations isolated from ventral or dorsal trachea, respectively, cultured in air-liquid-interface (ALI) in control or RAR antagonist (BMS493) …
Figure 4—figure supplement 1
RAR signaling is active in ventral and dorsal luminal cells but not in basal cells (BCs) of the adult tracheal epithelium during homeostasis.
(A–C) X-gal staining of adult trachea (tr), thyroid (thy) and esophagus (eso) from RARE-LacZ reporter mice showing strong LacZ expression in the tracheal and thyroid cells (positive control) but not …
Figure 5 with 3 supplements
BC-2 and BC-1 have distinct behaviors during initiation of repair of the damaged epithelium in mouse models of injury.
(A) Diagram: strategy for induction of airway injury by Naphthalene (Nap, intraperitoneal) or Polidocanol (Poli, intratracheal) and analysis at 5dpi post-Nap or 7dpi post-Poli. (B–C) IF of tracheal …
Figure 5—figure supplement 1
Distinct kinetics of repopulation between the ventral and dorsal BC-derived tracheal epithelium post-Naphthalene (5dpi) and post-Polidocanol (7dpi) injury in adult mice.
IF of representative areas showing ventral and dorsal distribution of markers of secretory (SCGB3A2), multiciliated (FOXJ1), BC (TRP63, KRT5), proliferation (KI67) and metaplastic (KRT13) cells. …
Figure 5—figure supplement 2
BC-1 and BC-2 markers maintain their differential ventral-dorsal enrichment in basal cells during repopulation of the airway epithelium post-injury.
Immunofluorescence of KRT5, a-SMA, BC-1 (TGM2, ISL1) and BC-2 (KRT17, CAV1, and CD44) markers in ventral and dorsal tracheal epithelium of 5dpi-Naphthalene or 7dpi-Polidocanol. Small panels are …
Figure 5—figure supplement 3
Regional differences in ventral-dorsal programs of repopulation are no longer evident at late stages of regeneration post injury.
(A–B) IF of KRT5 and CC10 in ventral and dorsal tracheal epithelium of 15dpi-Naphthalene or 15dpi-Polidocanol. (C–D) Morphometric analysis of KRT13 + cells in the ventral or dorsal adult tracheal …
Figure 6
Basal cell heterogeneity is established during embryonic development.
(A–B) IF of BC-1 and BC-2 markers co-labeled with KRT5 in tracheal sections from E18.5 WT and Foxg1Cre; Sox9fl/fl littermates. BC-1 and BC-2 markers maintain the ventral-dorsal differential …
Figure 7
Airway basal cell spatial heterogeneity is conserved in human airways.
(A) IF of ISL1, CD44, CAV1 and KRT17 co-stained with KRT5 in adult human tracheal sections. Quantification of fluorescence intensity in ventral and dorsal BCs from paired regionally distinct areas. …
