Figure 1. Single cell RNA sequencing of mouse PNECs reveals expression of dozens of peptidergic genes.

(A) Strategy for labeling, enrichment, and scRNA-seq of exceedingly rare PNECs. Timeline (top) of tamoxifen (Tam) injections (gray arrowheads) of Ascl1^CreER/+;Rosa26^{LSL-ZsGreen/+} mice beginning at embryonic day (E) 13 - E14 to permanently induce ZsGreen expression in pulmonary neuroendocrine cells (PNECs or NE cells). Lungs were dissected at indicated ages (black arrowheads, postnatal day (PN) 21, PN90, and PN120) and mechanically and enzymatically dissociated (dissoc.) into single cells. Endothelial (CD31+) and immune (CD45+) cells were depleted by magnetic-cell sorting (MACS) then PNECs enriched by fluorescence-activated cell sorting (FACS) EpCAM⁺/Zsgreen⁺ double-positive cells. Sorted cells were analyzed by plate-based scRNA-seq using SmartSeq2 protocol. (B) Most sensitive and specific PNEC markers identified by scRNA-seq, ranked by ratio of the natural logs of the average expression (ln (counts per million, CPM + 1)) of the marker in PNECs (NE cells) vs. non-PNEC (non-NE) lung epithelial cells in mouse lung cell atlas (Travaglini et al., 2020). *, previously known PNEC marker. (C) Violin plots showing expression of five new markers (Resp18, Pcsk1, Scg5, Chgb, Sez6l2) and three previously known markers (*; Calca, Syp, Chga) across 40 cell types from mouse lung cell atlas (Travaglini et al., 2020). From left to right (x-axis): (1) neuroendocrine (NE, PNEC), (2) club, (3) multiciliated, (4) basal, (5) goblet, (6) alveolar type 1, (7) alveolar type 2, (8) glial, (9) smooth muscle, (10) myofibroblast, (11) adventitial fibroblast, (12) alveolar fibroblast, (13) pericyte, (14) mesothelial, (15) chondrocyte, (16) artery, (17) vein, (18) capillary aerocyte, (19) capillary-general, (20) lymphatic, (21) B cells, (22) Zbtb32+ Bcells, (23) plasma, (24) CD8+ T, (25) CD4+ T, (26) regulatory T, (27) Ly6g5bt + T, (28) natural killer, (29) Alox5+ lymphocyte, (30) neutrophil, (31) basophil, (32) alveolar macrophage, (33) interstitial macrophage, (34) plasmacytoid dendritic, (35) myeloid dendritic type 1, (36) myeloid dendritic type 2, (37) Ccr7+ dendritic, (38) classic monocyte, (39) nonclassical monocyte, (40) intermediate monocyte. (D) Close-up of neuroepithelial body (NEB) in PN155 wild type (C57BL/6NJ) mouse lung probed by multiplex single molecule RNA fluorescence in situ hybridization (smFISH) to detect expression of indicated PNEC markers, with DAPI nuclear counterstain. Dashed circles, individual PNECs (numbered); dashed line (basement membrane). Scale bar, 10 μm. Quantification (right) of clustered PNECs that express indicated markers (n=76 cells scored in left lobe and right lower lobe). Note classic marker Calca (CGRP) was not detected in 6% of Resp18⁺Scg5⁺double-positive PNECs. (E) Quantification of peptidergic gene expression in PNECs by scRNA-seq. Bars show percent of profiled PNECs (NE cells, n=176) with detected expression of the 43 peptidergic genes indicated; values above bars are log-transformed mean gene expression (ln (CPM + 1)) among expressing cells. Black dots, expression values from a second PNEC dataset (filled circles, n=92 PNECs) in which PNECs were genetically labeled using Cgrp^CreER;Rosa26^LSL-ZsGreen mice, sorted, and isolated on a microfluidic platform (Ouadah et al., 2019). (Comparison by Fisher’s exact test (two-tailed) of the proportions of PNECs detected in the two scRNA-seq datasets is provided in Supplementary file 4, with caveat that the comparison is of results from different techniques on different samples.) *, Previously known mouse PNEC peptidergic genes; blue highlight, classic hormone genes. (F) Micrograph of NEB from PN90 Ascl1^CreER/+;Rosa26^{LSL-ZsGreen/+} mouse lung immunostained for CGRP and newly identified PNEC neuropeptide CARTPT. White arrowheads, CGRP⁺ PNECs; red arrowheads, CARTPT⁺ PNECs; *, CGRP^- CARTPT^- double-negative PNEC. Right panel, quantification of CGRP and CARTPT staining in PNECs defined by Ascl1-CreER-lineage label (n=237 PNECs scored in three PN60 Ascl1^CreER/+;Rosa26^{LSL-ZsGreen/+} mice). (G) Quantification of PNEC (NE cell) expression of the indicated peptidergic genes by scRNA-seq (black bars, n=176 cells) and multiplex smFISH (grey bars and dashed extensions, n=100cells scored in NEBs from 2 mice, see Figure 2). Grey bars, cells with high expression (>5 puncta/cell); dashed extensions, cells with low expression (1–4 puncta/cell). Fisher’s exact test (two-tailed) gave p=1 (not significant) for all comparisons of proportions of expressing PNECs for each gene as detected by smFISH (>5 puncta/cell) vs. scRNA-seq (black bars); when the comparisons included cells with 1–4 puncta/cell by smFISH, differences were significant (p<0.05) for Chga, Cartpt, Agt, Pomc, Nmb, and Adcyap1 but not for Scg5 and Calca (p=0.9 for both).

Figure 1—figure supplement 1. FACS enrichment strategy for PNECs.

(A) Fluorescence-Activated Cell Sorting (FACS) gating path of adult (PN120) mouse lung cells with PNECs genetically lineage-labeled with ZsGreen and with immune (CD45+) and endothelial cells (CD31+) depleted by MACS prior to flow sorting as shown in Figure 1A. Red boxes, cell population selected at each gate; percentage values, percent of cells within the indicated gate. Gate 1, side scatter (SSC) and forward scatter (FSC) to separate cells (from debris). Gate 2, forward scatter width (FSC-W) and height (FSC-H) to separate single cells (from doublets and larger aggregates). Gate 3, DAPI to select viable cells, and APC to exclude endothelial (CD31+) and immune (CD45+) cells. Gate 4 (Q2),ZsGreen+ (Ascl1 genetic lineage label) and EpCAM+ (epithelial) to enrich for neuroendocrine (NE) cells; alternative gates were also used at this step to sort control cells (Q1, ZsGreen^loEpCAM^hi; Q4, ZsGreen^lo EpCAM^hi). (B) Results of an analytic sort of 100,000cells using above scheme. Count, number of cells within each gate. % of parent, % of analyzed cells at that sort; % of total, % of total analyzed cells (100,000). (C) Computational clustering using t-distributed stochastic neighbor embedding (t-SNE) of the expression profiles of 534 experimental and control cells sorted as above and analyzed by scRNA-seq. Identity of the cells (dots) in each cluster (colors) was assigned by cluster-selective expression of the canonical lung cell type markers indicated. (C’) Box and whisker plots comparing Epcam RNA levels (determined by scRNA-seq) in PNECs obtained from sorted ZsGreen^hi EpCAM^hi (left) vs. ZsGreen^hi EpCAM^lo (right) cell populations. Note EpCam RNA levels in PNECs obtained from both sorted populations are indistinguishable. (C'') t-SNE clustering of the 176 PNECs in the boxed region of panel C highlighting PNECs obtained from the ZsGreen^hi EpCAM^hi (pink dots) vs. ZsGreen^hi EpCAM^lo (teal dots) sorted populations. Note intermingling of PNECs from the two sorted populations. (D) Number and fraction of cells obtained by each gating strategy. NE, neuroendocrine (PNECs); AT2, alveolar type 2; AT1, alveolar type 1; Endo, endothelial. (E) Scatterplot showing sensitivity (% NE cells that express the marker) vs. specificity (% non-NE epithelial cells that express the marker) of a panel of PNEC markers by scRNA-seq. Among the most sensitive and specific genes, several genes are likely also PNEC-selective (indicated by asterisk). The most PNEC-selective genes are also indicated. Classic PNEC genes (bold font). NE, neuroendocrine. (F) Violin plots comparing expression of representative top marker genes in common (PNEC and TNEC markers) between pulmonary neuroendocrine cells (PNECs) and tracheal neuroendocrine cells (TNECs), and the TNEC-selective and PNEC-selective markers (Supplementary file 1). The 40 lung cell types in the mouse lung cell atlas are indicated along x-axis (left to right) as in Figure 1C, beginning with PNECs (orange dot) at left. Pulmonary epithelial cells (Pulm epi.) indicated along x-axis. TNEC expression of each gene is compared to other tracheal epithelial cells (Trach. epi.); data from Montoro et al., 2018. The 6 tracheal epithelial cell types along the x-axis (left to right) are listed in the following order: TNEC (blue dot), basal, club, ciliated, tuft, and ionocyte. Expression of tracheal cells reported as log-normalized unique molecular identifiers per 10,000 (ln (UPK+1)), y-axis.