Abstract
Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of ‘squamous metaplasia’, which is long thought to be a precursor of lung cancer.
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Data availability
Plate-based single-cell sequencing data have been deposited to the GEO: GSE207479. 10X-based single-cell sequencing data have been deposited to the GEO: GSE244215. Bulk RNA and ATAC sequencing of mouse hillock and pseudostratified basal cells have been deposited to the GEO: GSE253021 (RNA) and GSE253020 (ATAC). High-resolution images of human wholemounts have been deposited to the Harvard Dataverse at https://doi.org/10.7910/DVN/6JDXOP62. The KRT13-CreER mouse line has been deposited at Jax (#038257). Source data are provided with this paper.
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Acknowledgements
We thank the members of the Rajagopal laboratory for critical review of the manuscript and discussions; and New England Donor Services (NEDS) for providing human airway samples and the generosity of the patients who agreed to donate their tissue to science. Electron microscopy was performed in the Microscopy Core of the Program in Membrane Biology, which is partially supported by the Inflammatory Bowel Disease grant DK043351 and the Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK057521. We thank J. Griffith for providing influenza virus-expressing GFP; the Single Cell Core and the BPF Genomics Core Facility (RRID: SCR_007175) at Harvard Medical School for performing the barcoding and library preparation for 10X single-cell RNA sequencing; and K. Po Tse for cryosectioning expertise. Funding was provided by the Cystic Fibrosis Foundation (LIN19F0) to B.L.; the US National Institutes of Health (5T32HL 116275-9 and 5U24HL148865-04/OS00000379) and the Cystic Fibrosis Foundation (CFF003338L121) to V.S.S.; the National Institutes of Health (F31HL165736) to C.C.; the National Institutes of Health (K08HL124298-05) to V.V.; the National Institutes of Health (UG3CA268117 and UH3CA268117) to Z.D. and J.R.; and the National Institutes of Health (R01HL118185-08, R01HL157221-01 and R01HL142559-04) and the Bernard and Mildred Kayden Endowed MGH Research Institute Chair to J.R.
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B.L., V.S.S., C.C., V.V., A.W. and J.R. conceptualized the study. B.L., V.S.S., C.C., V.V., A.W., J.X., G.G.S., M.V.S., A.L., Y.X., J.S., C.A.H., D.E.C., J.V. and L.P.H. conceived the methodology and performed the investigation. B.L., V.S.S. and C.C. designed, carried out and analysed the majority of the experiments. D.E.C. conducted electron microscopy. J.S. performed the basal cell culture experiments. V.V. and J.V. conducted live, two-photon imaging and the majority of the naphthalene injury experiments. G.G.S., Y.X. and A.L. designed, conducted and analysed the hillock-optimized dissociation single-cell RNA sequencing. A.W. established the initial human tissue collection. J.X. conducted the rare-cell experiments. C.A.H. conducted the immunostaining of sectioned trachea. J.S. and M.V.S. conducted the influenza experiments. B.L., V.S.S., C.C., V.V., Z.D. and J.R. acquired funding. J.R. administrated the project. B.L., L.P.H. and J.R. supervised the study. B.L., V.V., L.P.H. and J.R. wrote the original draft of the manuscript. B.L., V.S.S., C.C., V.V., A.W., J.X., M.V.S., J.S., G.G.S., Z.D., L.P.H. and J.R. reviewed and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Phenotypic and molecular characterization of hillocks.
A, Schematic of wholemount preparation. B, Representative wholemount images of inbred C57Bl6, CD1, and outbred C57Bl6 mice (primary strain of mice used in this manuscript) showing variability of the characteristic pattern of hillock size and location. C, Quantification of the percent tracheal surface area covered by hillocks in a variety of murine backgrounds at 10-12 weeks of age. Each dot represents a biologically independent mouse replicate. SD is marked. D, Quantification of hillock size in 16 week old male and female mice on a mixed inbred background show no significant sex-dependent difference. Each dot represents a single wholemount trachea. (Female: n = 6, male: n = 5, Shapiro-Wilk normality test p = 0.120, equal variance test p = 0.774, two-tailed t-test p = 0.441). Error bars are SD and mean is shown. E, Representative transmission electron microscopy panels of the interface of two hillock cells showing interdigitation between the dotted lines. F, Quantification of rare cells in hillocks (n = 3 biologically independent animals). Mean is marked.G, UMAP representation of single cell RNAseq profiles obtained using the SMART-seq protocol on microdissected regions of trachea. Black dots represent basal cells isolated from pseudostratified epithelium surrounding putative hillocks. Red dots represent cells isolated from microdissected putative hillock patches of tamoxifen induced TRP63-CreER;LSL-TdTomato mice. H, Unbiased cluster assignment of single cell data, separates hillock luminal cells, hillock basal cells, and pseudostratified basal cells. Cells isolated from microdissected hillock patches are enriched for hillock luminal and basal cells. (I, Violin plots demonstrating that both hillock basal and luminal cell clusters specifically express Krt13, Krt6a, and Dsg3. Basal hillock cells additionally express Trp63 like their pseudostratified counterparts. Hillock luminal cells uniquely express Serpinb2. Basal cells of pseudostratified epithelium express Trp63 but not the hillock cytokeratins. J, Wholemount immunostaining of DSG3 puncta. K, Cryosection immunostaining of SERPINB2 and TRP63 within hillocks. Stain replicated on three trachea. Scale bar 20 µm (B), 300 nm (E), 50 µm (J), 10 µm (K).
Extended Data Fig. 2 Hillocks are resistant to papain dissociation.
A, Representative wholemount image of a tamoxifen induced TRP63-CreER;LSL-TdTomato trachea before (left panel) and after papain dissociation (right panels). Arrows point to regions of the trachea that are KRT13 + , which are the only regions of the epithelium that have resisted dissociation. B, Quantification of the percent of TdTomato signal remaining within hillocks vs surrounding pseudostratified epithelium. Each dot represents a single, biologically independent wholemount trachea. SD and mean are marked. C, Matrix of dissociation conditions and their efficacy in dissociating hillocks into a single cell suspension. Only papain followed sequentially by an enzyme mix effectively dissociates hillocks to yield single cell suspensions. Scale bar 250 µm (A, C).
Extended Data Fig. 3 Single-cell sequencing of hillock-optimized dissociated trachea.
A, UMAP representation of single-cell RNA-seq data from four trachea dissociated using an optimized enzymatic cocktail. B, Differential gene expression (DGE) analysis of hillock basal cells compared to all pseudostratified cells. C, DGE analysis of hillock luminal cells compared to all pseudostratified cells. D, Direct DGE comparison of hillock basal versus hillock luminal cells. E, Direct DGE comparison of hillock luminal cells versus secretory cells. In all DGE plots, yellow highlighted genes were identified in the previous SMART-seq based single-cell RNA-seq, and blue highlighted genes are unique adhesion-associated genes found using this new dataset.
Extended Data Fig. 4 Generation of a KRT13-CreER driver and demonstration that hillock luminal cells are not secretory cells.
A, Schematic of 2A-CreER cassette knocked into the native KRT13 locus via CRISPR-Cas9. B, Representative wholemount stains with high-mag insets (below) of KRT13-CreER and KRT6-CreER lineage trace mice 3 days post-tamoxifen induction showing high hillock cell labeling specificity. C, Representative wholemount stains of control KRT13-CreER and KRT6-CreER lineage trace trachea show no appreciable labelling in the absence of tamoxifen after 3 months. D, Quantification of efficiency and specificity of the two hillock-specific drivers with either one or two doses of tamoxifen. Left, efficiency was measured using cryosections and manual counting of cells that were labeled using either KRT13-CreER or KRT6-CreER mice. 3 animals for each condition were sectioned, and each dot represents a single section (KRT13 1x dose: n = 13, KRT13 2x dose: n = 3, KRT6: n = 5). Right, specificity was measured using confocal Z-slices through the entire airway. Each dot represents a single animal wholemount (KRT13 1x dose: n = 4, KRT13 2x dose: n = 3, KRT6: n = 3). E, A representative cryosection of a hillock in an unperturbed wildtype animal shows no UEA1+ goblet cells within the hillock. F, A representative image of a cultured wholemount explant treated with 20 ng/ml of IL13 and dual-SMAD (10uM A8301 and 10uM DMH1) inhibition. Secretory cells outside of the hillock are now UEA1 + , while hillocks remain UEA1 negative. G, Hillock luminal cells sorted on the basis of KRT13-CreER;TdT+ and GSib4- do not dedifferentiate and expand following dual-SMAD inhibition. Experiment replicated on three trachea. SD and mean are shown. Scale bar 250 µm (top of B, C), 50 µm (bottom of B) and F, 10 µm (E).
Extended Data Fig. 5 Hillocks harbour a pool of active stem cells that continually generate pseudostratified epithelium during homeostasis.
A, Clones of basal cells following a 3-month chase using the pan-basal TRP63-CreER driver. Asterisks mark two large clones in the hillock (left). Arrows mark single or two cell clones in pseudostratified ciliated epithelium (right). B, Representative wholemount images of KRT13-CreER mice 2 days, 1 month, and 3 months after lineage labelling of homeostatic trachea. Boxes represent the insets in main Fig. 1h. C, Representative confocal images of cryosectioned trachea 2 days post-labelling shown in B, stained for the basal cell marker TRP63 and the hillock marker KRT13. D, Representative wholemount images of KRT6A-CreER mice 3 months after lineage labelling along with a high-mag image below. E, Representative cryosections of the 3 month-long chase trachea shown in Fig. 1h that was initially stained as a wholemount for basal stem cells (KRT5), secretory cells (CC10), and ciliated cells (Atub). Arrowheads mark lineage traced basal, secretory, and ciliated cells. F, Quantification of the number of basal, secretory, and ciliated cells that are KRT13-CreER lineage labelled following a 3 month chase. Each dot represents data from a single sagittal section of the trachea counted in its entirety, evenly sampling 3 different animals. SD and mean are marked. G, Summary schematic of a hillock flanked by sparsely ciliated transition zones and surrounded by pseudostratified epithelium. Scale bars 100 µm (A, bottom of D), 250 µm (B, top of D), 20 µm (C), 10 µm (E).
Extended Data Fig. 6 Characterization of naphthalene injury.
A, Quantification of cell death after 80 mM HCl demonstrates decreased hillock cell death. B, Quantification of EdU+ nuclei after 80 mM HCl reveals increased hillock cell proliferation. C, Quantification of cell death after freeze-thaw with less death in the hillock. D, Quantification of EdU+ replicating cells after freeze-thaw with more replication in the hillock. E, Quantification of the percent area expressing influenza nuclear protein in pseudostratified vs hillock regions showing hillock resistance to flu infection. Each individual point represents a separate mouse trachea. Unpaired two-tailed t-tests were performed. Error bars represent SD, and mean is shown. Each dot represents an individual biological animal replicate. F, Naphthalene injury results in variable injury, representative images shown here. G, Quantification of the correlation of epithelial denudation with percent weight loss. Each dot represents a quantification based on a single wholemount trachea. H, Experimental schematic for Fig. 2d. I, Representative wholemount image of a trachea 3 days after naphthalene injury showing survival of KRT5/KRT13 double positive hillock regions. White box in top left demarcates KRT13- glands. J, Representative image of a hillock region (top) versus gland region (bottom) showing that glands do not express KRT13 after injury. K, Representative confocal image of a section of the trachea showing KRT13+ Foxj1+ double positive cells. L, Quantification of the percent total surface area of the trachea that is KRT13+ after naphthalene injury. Each dot represents a biologically independent mouse replicate. SD and mean are marked. Two-tailed unpaired t-tests were performed. Scale bars 250 µm (F,I), 20 µm (J), 10 µm (K).
Extended Data Fig. 7 Hillocks regenerate the tissue after naphthalene injury.
A, Representative wholemount images of control trachea subjected to naphthalene injury in the absence of tamoxifen induction show minimal lineage labelling. B, High-mag images showing lack of leakiness following naphthalene exposure when tamoxifen is not administered despite high levels of KRT13 or KRT6A expression. Exceedingly rare labelled cell is shown as evidence of label detection. C, Quantification of labelled and unlabelled basal, ciliated, secretory, tuft, neuroendocrine, and ionocyte cells in KRT13-CreER;LSL-TdTomato lineage traced animals after naphthalene. No pairwise comparisons were statistically different from each other (1-way ANOVA). Each dot represents a cryosection quantified in a 20x FOV, spread across three distinct biologically independent replicates. SD and mean are marked. D, Representative KRT13-CreER ifgMosaic clonal lineage trace wholemounts after naphthalene injury showing large, single-clones contributing to tracheal regeneration. E, Representative immunostaining of sections taken from clonal KRT13-CreER trachea after naphthalene injury, showing all cell lineages are represented and that clones contain multiple lineages. Scale bars 250 µm (A), 10 µm (B, E), 500 µm (D).
Extended Data Fig. 8 Bulk RNA and ATAC-seq analysis of cultured mouse hillock and pseudostratified basal stem cells.
A, Gating strategy and representative FACS plot for hillock basal cell versus pseudostratified basal cell sort from murine trachea. Hillock basal cells: EpCAM-APCCy7 + , p63GFP + ,tdTomato + ; pseudostratified basal cells: EpCAM-APCCy7 + , p63GFP + , tdTomato-. Percentages of each gate with respect to that panel and of the parent population are shown. Live cells were initially sorted on epithelial marker EpCAM, then p63 and hillock lineage reporter. B, Analysis of ATAC-seq data showing that cultured hillock basal cells exhibit greater chromatin accessibility throughout the genome (top) and specifically at transcription start sites (middle and bottom). C, Representative tracks of ATAC reads surrounding the Etv4, Cyp26b1 and Flna gene loci showing increased accessibility in hillock basal cells. D, Volcano plot of all differentially expressed genes, cultured mouse hillock basal stem cells (right) versus pseudostratified basal stem cells (left). E, GO analysis of DEGs (C) highlights an enrichment of pathways associated with focal adhesions and cell junctions. F, Volcano plot of ECM and cell-cell junction genes demonstrates a selective enrichment of integrins and junctional proteins in hillock basal cells (green) versus pseudostratified basal cells (magenta).
Extended Data Fig. 9 Hillock basal cells respond to retinoic acid signalling inhibition by generating injury-resistant hillock structures that display features of keratinizing squamous metaplasia.
A, 10X low mag (left) and 25x high mag (right) image of an air-liquid interface (ALI) epithelial culture derived from KRT13-CreER labeled hillock basal cells and lineage negative pseudostratified basal cells cultured in standard ALI conditions. Arrows mark lineage-labelled ciliated cells. B, 10X low mag (left) and 25x high mag (right) image of an ALI culture derived from KRT13-CreER labelled hillock basal cells and lineage negative pseudostratified basal cells cultured with Agn193109 showing that hillock-derived epithelium is aciliate while unlabeled pseudostratified basal cells develop ciliated cells. C, Quantification of area covered by hillock basal cell-derived cells shows that Agn193109 treatment induces significantly higher coverage of the ALI by hillock-lineage traced cells. D, Optical slice reconstruction (60X) of ALIs seen in B. Brackets demarcate the thickness of the epithelium. E, Low magnification image of ALI cultures subjected to 20 min of trypsin dissociation condition. F, Quantification of nuclei remaining after trypsin dissociation shows Agn193109 treated cultures resist dissociation. G, Microscopy of ALI cultures infected with influenza and stained for influenza nucleoprotein (NP) shows that KRT13+ patches that expand after Agn193109 treatment selectively resist infection. H, Transepithelial electrical resistance (TEER) measurements of ALI cultures shows significantly higher TEER after Agn193109 treatment. I, Representative cryosections of purified pseudostratified vs hillock basal cells-derived ALI cultures in standard differentiation media. Scale bars 500 µm (left panels of A and B, E), 100 µm (right panels of A, and B), 20 µm (D, I), 250 µm (G).
Extended Data Fig. 10 Human hillocks exist.
A, Image of an entire airway tree (donor HU76) stained for KRT13 using horseradish peroxidase and DAB substrate demonstrates the presence of KRT13+ patches of epithelium. B, Images of the airway trees of donor HU72 and HU75 demonstrating aciliate, KRT13+ patches of epithelium marked by brown DAB stain. On HU75, Atub is additionally identified using alkaline phosphatase (red). C, Insets of the magenta-boxed regions in A and B representing patchy KRT13 expression found outside of the characteristic locations of hillocks which do not possess the characteristic morphology associated with murine hillocks. D, Section of a prospectively identified and subsequently microdissected KRT13+ hillock patch demonstrates the characteristic histologic pattern associated with murine hillocks. E, The same immunofluorescent image shown in Fig. 5g, counterstained with DAPI. Expanded insets in the lower panels highlighting the ciliated, transition, and hillock zones. Arrowheads mark squamous flat cells. F, Representative cryosection of a normal human ciliated epithelium exhibiting scattered suprabasal KRT13+ cells marked by arrows. G, TEER of human ALIs derived from ciliated versus hillock basal stem cells, with and without AGN193109 treatment. 1-way ANOVA was performed, followed by Tukey’s multiple comparisons test. H, Increased TEER in hillock derived cultures. I–K, Quantification of ALI cultures of hillock and pseudostratified epithelium after (I) influenza infection, (J) cryospray injury, (K) acid injury, demonstrating human hillock injury resistance. Unpaired two-tailed t-tests were performed. Each dot represents a distinct ALI membrane. SD and mean are marked. Scale bars, 1 cm (A, B), 50 µm (D–F).
Supplementary information
Supplementary Information
This file contains Supplementary Tables 1-2 and legends for Supplementary Videos
Supplementary Video 1
3D rendered movie of a single hillock cell visualized using photoactivatable GFP. Basement membrane is shown in white, detected through second harmonic generation signal. Rotation reveals that the hillock cell does not touch the basement membrane
Supplementary Video 2
3D rendered movie of two hillock cells visualized using photoactivatable GFP. Basement membrane is shown in white, detected through second harmonic generation signal. Rotation reveals that neither hillock cell touches the basement membrane
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Lin, B., Shah, V.S., Chernoff, C. et al. Airway hillocks are injury-resistant reservoirs of unique plastic stem cells. Nature (2024). https://doi.org/10.1038/s41586-024-07377-1
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DOI: https://doi.org/10.1038/s41586-024-07377-1
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