A molecular circuit regulates fate plasticity in emerging and adult AT2 cells

Abstract

Alveolar Type 1 and Type 2 cells are vital for lung gas exchange and become compromised in several diseases. While key differentiation signals are known, their emergence and fate plasticity are unclear. Here we show in the embryonic lung that single AT2s emerge at intermediate zones, extrude, and connect with nearby epithelium via interlumenal junctioning. We observe AT2s retain fate plasticity until the bZIP transcription factor C/EBPα suppresses Notch signaling at a novel Dlk1 enhancer. Both Dlk1 and Cebpa are regulated by the polycomb repressive complex (PRC2), which together form a "pulse generator" circuit that times Dlk1 expression and thus Notch activation, resulting in a "salt and pepper" pattern of AT1 and AT2 fate. In injured adult lungs, C/EBPα downregulation is required to re-access AT2 fate plasticity and is mediated by the dominant negative C/EBP family member CHOP. Finally, Cebpa loss also activates a "defender" AT2 state, distinct from its reparative state, and we propose AT2s toggle between either state following infection to protect and repair alveoli.

Fig. 1. Emergence and patterning of nascent AT2 cells.

a scRNAseq UMAP generated from a published timecourse study (GSE119228). b Leiden clustering identifies mature AT2s (mAT2), nascent AT2s (nAT2), distal progenitors (DP), and AT1s. c Cluster proportion of AT2 lineage across development shows their separation over time. d Venn diagram indicates the number of genes shared or restrictively expressed. e Dot plot of stage-restricted marker gene Retnla (nAT2) and Cd74 (mAT2). f E17.5 immunostain of RELMα (green), CD74 (red), and DP/AT1-restrictive luminal marker PDPN (white). Bar, 25 µm. g E15.5 (left) and E16.5 (right) immunostain for smooth muscle actin (SMA, red), RELMα (green), E-cadherin (ECAD, blue), and PDPN (white). Close-up images for E15.5 lungs of rare single nAT2s (asterisk) emerging at intermediate regions, not terminal end buds (arrowheads). Bars, 100 µm (left and right panels), 20 µm (close-ups). Mesothelium is marked with white dash. h Quantification of RELMα⁺ nAT2 immunostains from E15.5 to E17.5 (n represents branches scored per timepoint; data are represented as median with whiskers extending to the upper and lower quartiles. p values determined using Kruskal–Wallis test). i Quantification of RELMα⁺ nAT2 morphology (columnar versus basally extruded). Extruded values presented as mean ± SD (n represents cells scored at each timepoint). j Schematic of the timing and sequence of nAT2 emergence. At E15.5 the first nAT2 emergent zone (bright green) occurs between the terminal end bud and stalks of the undifferentiated distal epithelium (blue), which ~E16.5 expands during branching morphogenesis. Around E17.5, nAT2s next emerge more proximally (dark green) as airway smooth muscle remodels into myofibroblasts—leaving the final emergence of nAT2s to occur at the distal tips after birth. Source data are provided as a Source Data file.

Fig. 2. Nascent AT2s form junctions with nearby but anatomically distinct lumens.

a E17.5 immunostain of RELMα (green), PDPN (white), and DAPI (blue) depicting where nAT2s are observed traversing the interstitium to form interlumenal junctions (ILJs). Magnified confocal sections (yellow box, i) of a representative nAT2 cell embedded in lumen A, at z-position +18 µm (top right), as well as in lumen B, at z-position +24 µm (bottom right). Bar, 50 µm (left), 10 µm (close-ups; right). b Quantification of ILJs during development (n represents nAT2 cells scored at each timepoint). ****p =  4.8 × 10⁻¹¹ (one-way ANOVA, data as mean ± SD). c 3D rendering immunostaining of a direct (upper, AT2-to-AT1) and indirect (lower, AT2-to-AT2) ILJ from an immunostain for E-cadherin (green), RELMα (red), PDPN (white), and DAPI. Lumen boundaries are marked in yellow dashes. d Schematics of approach for timelapse confocal microscopy of Nkx2.1^Cre; Rosa26^mTmG lineage labeled precision cut lung slices (Adapted from BioRender) depicted in (e, f). e Representative direct nAT2 junctions between two lumens in 12-h timeperiod. f Two nAT2s established contact at 14.5 h and junction between two lumens in 15-h snapshots, contact marked with white arrows. g Representative MUC1 domain length in non-junctioning (left) and junctioning nAT2s. h Quantification of (g) shows a reduction in MUC1 domain length following ILJ (n represents number of nAT2 cells scored at each timepoint). ****p =  4.8 × 10⁻¹¹ (Student’s two-sided t test, data as mean ± SD). i Observed sequence of nAT2 basal extrusion and apical constriction. j Schematic of junctioning outcomes (none, indirect, or direct) as interstitial thickness decreases. Source data are provided as a Source Data file. Created in BioRender. Cai, J. (2025) https://BioRender.com/9wf54yt.

Fig. 3. Nascent AT2s retain fate plasticity.

a PN1 and adult (≥PN60) lungs immunostained for AT2 marker MUC1 (white), RELMα (green), CD74 (red), and DAPI. Bar, 10 µm. b Timecourse quantification of the nAT2 transition to mAT2. p values determined using Brown–Forsythe and Welch ANOVA test (n represents AT2s scored per timepoint in experimental triplicate, values are mean ± SD). c PN1 immunostains for RELMα (green), CD74 (red), and DAPI, showing the pattern of mAT2s and nAT2s. Bar, 50 µm. d Schematic contrasting the reported proximal-distal pattern of AT1/AT2 differentiation with the sporadic pattern of AT2 maturation. e scRNAseq PAGA velocity analysis of E15.5 and E17.5 distal lung epithelial cells. Arrow thickness represents the relative cell fraction along a depicted cluster trajectory. A trajectory is observed from nAT2s to nAT1s (red arrow). f Velocity magnitude and AT1/AT2 gene score for the nAT2 fraction from (e). g Velocity analysis of the nAT2 fraction from e depicting AT1/AT2 score (cutoff 0.5). h UMAP of scRNAseq timecourse dataset (GSE149563) of the distal epithelium. i Leiden clustering identifies the AT2-to-AT1 transitional cluster (tAT2 > 1; brown) whose AT1 trajectory is confirmed by velocity analysis (red box). j Timepoint distribution within the tAT2 > 1 cluster. k Representative organoids cultured on Matrigel for 4 days in the presence of FGF7 (50 ng/mL), derived from either E15.5 DPs, E18.5 nAT2s, PN3 mAT2s, or adult mAT2s. AT2 and AT1 fate is detected by immunostaining for PDPN (white), RAGE (red), DAPI (blue), and AT2 marker SFTPC (green). DPs express both the AT1 (RAGE, PDPN) and AT2 (SFTPC) markers. Bars, 20 µm. l Quantification of (k) cell percentage per spheroid that differentiated into AT1. Data are represented as box plots showing median, upper, and lower quartiles, and the whiskers extend to the minima and maxima. p values determined using Kruskal–Wallis test. (n = spheroids per condition pooled from 3 independent experiments. m A revised model alveolar epithelial differentiation wherein a window of fate plasticity exists during which AT2s retain the ability to rapidly differentiate into AT1s. All experiments were repeated at least three times. Source data are provided as a Source Data file.

Fig. 4. C/EBPα a is required to maintain but not select AT2 fate.

a Bioinformatic screen of 1678 transcriptional or epigenetic regulators, of which 67 are enriched along AT2 lineage. 32 of these have known embryonic lethal or lung defects upon knockout in mice and can be stratified into four temporal categories: DP, DP-nAT2, nAT2-mAT2, and constitutive. The observed plasticity window is depicted in red. b Dot plot of Sox9 (DP restrictive; yellow), Cebpa and Nupr1 (nAT2-mAT2 restrictive; pink), Etv5 and Elf5 (constitutive; gray) temporal expression (plasticity window in red). c E16.5 and E17.5 lungs immunostained for PDPN (white), C/EBPα (green), ECAD (red), and DAPI (blue). C/EBPα⁺ cells are marked (arrowheads) within the proximal (P) and distal (D) regions. Bars, 100 µm. d Alveolar regions of PN0 control Nkx2.1^Cre; Rosa26^mTmG; Cebpa^wt/fl (left) and Nkx2.1^Cre; Rosa26^mTmG; Cebpa^fl/fl (right) lungs immunostained for DP lineage reporter (GFP) and MUC1 (white). Note GFP⁺ AT2s and AT1s (flat and MUC1⁻, labeled “1”) form even after Cebpa deletion. Bars, 50 µm. e Alveolar regions of adult control LyzM^Cre; Rosa26^mTmG; Cebpa^wt/fl (left) and LyzM^Cre; Rosa26^mTmG; Cebpa^fl/fl (right) lungs immunostained for MUC1 (white) and CD74 (blue). AT2-lineage derived GFP⁺ cells differentiate into AT1s (asterisk) upon Cebpa deletion. Bars, 100 µm. f Quantification of (e) showing percent GFP⁺ cells that differentiate into AT1s upon Cebpa deletion (n represents number of GFP⁺ cells sampled for the Cebpa^wt/fl and Cebpa^fl/fl conditions in experimental triplicate). ****p =  1.4 × 10⁻¹⁴ (Student’s two-sided t test, data as mean ± SD). All experiments were repeated at least three times. Source data are provided as a Source Data file.

Fig. 5. C/EBPα blocks AT2 fate plasticity by repressing Dlk1.

a Design of scRNAseq experiment (upper). UMAP (i, lower left) with Louvain clustering depicts AT1s and AT2s (both control and Cebpa floxed) groups. Dot plot (ii, lower right) of gene score corresponding to cell type (AT1, AT2) or state (nAT2, mAT2) from all clusters. Note the change in scores following Cebpa deletion, suggesting some reacquisition of nAT2 state (red dashed box). b Design of iSuRe-Cre experiment (above). Representative immunostains for tdT-lineage (red) and EdU (green) from Cebpa deleted lungs with either high (~99% AT2s) or sparse dose (~20% AT2s) TMX (lower). Note lineage labeled cells that have proliferated (asterisk) or are AT1s (arrowhead). Bars, 50 µm. c Quantification of (b) showing percent tdT⁺ AT1s upon Cebpa deletion in either High Dose (fl/fl—H.D.) or Sparse (fl/fl—S) deletions compared to control (wt/fl). p values determined using One-way ANOVA with Tukey’s multiple comparisons testing (n represents tdT⁺ cells sampled for each condition in experimental triplicate; data as mean ± SD). d Screen for upregulated genes encoding a non-cell autonomous signal (left). Of the 107 genes upregulated following Cebpa deletion (log₂ FC ≥ 0.7), 22 encode extracellular proteins, 6 of which are expressed in nAT2s. The 4 genes that encode either a secreted (green) or membrane bound (red) protein are plotted by fold change (right). e Representative regions of control or Cebpa deleted mouse lungs immunostained for DLK1 (green) and tdT (red). Bars, 20 µm. f Quantification of (e) DLK1 expression by tdT⁺ AT2s in control, sparse, and high dose conditions. Note similar amounts in high dose and sparse deletion conditions, 47.2% and 43.9% respectively. p values determined using One-way ANOVA with Tukey’s multiple comparisons testing (n represents tdT⁺ cells sampled for each condition in experimental triplicate; data as mean ± SD). All experiments were repeated at least three times. g Bulk ATACseq tracks showing chromatin accessibility of Cebpa^wt/fl (blue) and Cebpa^fl/fl (red) AT2 cells (n = 3 replicates for each condition), showing the Dlk1 transcriptional start site and intergenic differentially methylated region (IG-DMR). The closest C/EBPα binding site to Dlk1 is observed at a distal trans-regulatory element (TRE, yellow) downstream of the IG-DMR, which displays reduced chromatin accessibility upon Cebpa loss. ChIP-seq of AT2s for C/EBPα shows increased binding at the putative TRE (red box). Source data are provided as a Source Data file.

Fig. 6. PRC2 and C/EBPα act as an incoherent FFL to generate a DLK1 pulse late in development.

a E15.5 (top) and E17.5 (bottom) lungs immunostained for DLK1 (green) and ECAD (red), close-ups show DLK1 intensity (fire LUT). Bars, 10 µm. b Expression of Cebpa, Dlk1, and PRC2 member genes (red) over time in distal lung epithelium. Note nominal Cebpa and Dlk1 expression before E16.5 (gray box). c E16.5 and E17.5 distal branches immunostained for PDPN (white), EZH2 (green), and ECAD (red) with distal tips indicated (D). Bars, 50 µm. d Immunostained E15.5 DPs differentiated with FGF7 alone without (Control) or with GSK126 (10 µM). Bars, 20 µm. e Quantification of (d) of DLK1⁺ cells per spheroid at day 4 (n represents spheroid number per condition in experimental triplicate). Data as mean ± SD (Student’s two-sided t test). f Cistrome analysis of the Cebpa locus identifies EZH2 (red) and the other PRC2 members (gray) with enriched binding. g E15.5 DP spheroids cultured as in (d) immunostained for C/EBPα. Bars, 20 µm. h Quantification of (g) percent C/EBPα⁺ cells per spheroid (n represents number of spheroids for each condition in experimental triplicate). Data as mean ± SD (Student’s two-sided t test). i Distal epithelial branch of an E15.5 Nkx2.1^Cre; Rosa26^mTmG; Ezh2^fl/fl mouse lung with sparsely labeled DPs (GFP⁺) wherein Ezh2 is deleted that is immunostained for C/EBPα (white). Bar, 10 µm. j Quantification of (i) percent lineage negative (RFP⁺) or lineage positive (GFP⁺) DPs expressing C/EBPα in the distal alveolar buds. Data are represented as box plots showing median, upper, and lower quartiles, and the whiskers extend to the minima and maxima. p values determined using two-tailed Mann–Whitney U-test (n represents number of distal alveolar buds sampled for both groups). k Proposed incoherent, type 2 Feed Forward Loop (I2-FFL) model. l Fitting of observed Dlk1 expression pattern from scRNAseq timecourse study to a mathematical I2-FFL Model (red line, RMSE ≤ 0.75 represents high goodness of fit). m E15.5 DP spheroids cultured as in (d), immunostained for PDPN (white), SFTPC (green), RAGE (red), and DAPI. Bars, 20 µm. n Quantification of (m) showing proportion of DPs (white), AT1s (gray) and AT2s (black). Data as mean ± SD (n represents number of spheroids per condition in experimental triplicate; one-way ANOVA). o Schematic of working model. A DLK1 pulse occurs upon loss of PRC2 in nAT2s, stimulating Notch signaling in DP or plastic neighboring cells. This pulse followed by lateral inhibition results in the observed pattern of AT1/AT2 fate. All experiments were repeated at least three times. Source data are provided as a Source Data file.

Fig. 7. C/EBPα downregulation is required to re-access mutually exclusive Defender and Regenerative states in response to injury.

a Experimental design for AAV Cebpa deletion study (top). Immunostained lungs 14 days post injury (bottom) showing GFP⁺ AT1s (asterisk). Bars, 100 µm. b Quantification of (a) percent GFP⁺ AT1s (n represents GFP⁺ cells sampled per condition in experimental triplicate). Data as mean ± SD (Student’s two-sided t test). c Adult mouse lung 16 days post Bleomycin injury immunostained for ECAD (green), C/EBPα (red), and DAPI (blue). Lower panel depicts C/EBPα expression (fire LUT) in injured versus uninjured sites. Bars, 100 µm. d Quantification of (c) mean C/EBPα intensity of AT2s within uninjured or injured regions. Data are represented as box plots showing median, upper, and lower quartiles, and the whiskers extend to the minima and maxima. p values determined using two-tailed Mann–Whitney U-test (n represents number of distal alveolar buds sampled per condition). e Dot plot of AT2s from published scRNAseq 1X Bleomycin timecourse study (GSE141259) depicting genes of known C/EBPα suppressors (Ddit3, Atf3, Atf4, and Ezh2—upregulation indicated in red) as well as DATP gene score. f Experimental design for stimulating CHOP in vivo. Control or CT020312 treated PN25 lungs immunostained for AT2 lineage (GFP) and MUC1 (white). Bars, 50 µm. g Quantification of (f) showing percent GFP⁺ AT1s in control and treated condition (n represents GFP⁺ cells sampled per condition). Data as mean ± SD (Student’s two-sided t test). h Leiden clustering identifies two Cebpa^fl/fl AT2 clusters: AT2^DLK1+ (green) and AT2^Def (red) which distinct velocity trajectories. i Gene Ontology (GO) analysis of the AT2^Def cluster (Fisher’s Exact Test). j UMAP with overlaid DATP and Defender gene scores. k SPRING plot of clusters in (h), showing AT2^Def state (red) in between the control AT2 (orange) and AT2^DLK1+ (green) clusters. l SPRING plot of the DATP-gene score. m Immunostained PN3 mAT2 spheroids cultured with FGF7 alone (Control, top) or with interferon-γ (20 ng/mL; bottom). Bars, 20 µm. n Quantification of (m) showing percent of AT1s per spheroid (n represents spheroids sampled per condition). Data as mean ± SD (Student’s two-sided t test). o Schematic working model of AT2s oscillating between AT2^Def (purple) and AT2^DATP (yellow) states, both suppressed by C/EBPα. Upon AT2 loss (gray AT2s) and C/EBPα downregulation following infection, remaining AT2s could oscillate (left) between either state allowing for both a proper response to infection and regeneration. In a static model (right), inability to access both AT2^Def and AT2^DATP states would result in either impaired pathogen response or repair, respectively. All experiments were repeated at least three times (adapted from BioRender). Source data are provided as a Source Data file.