Introduction

Alveoli are the sites of gas exchange in the lung that are lined by two distinct epithelial cell types. The flat surface of alveolar type 1 (AT1) cells facilitates gas exchange, while cuboidal alveolar type 2 (AT2) cells secrete surfactant to prevent alveolar collapse. Defective alveoli underlie several maladies—bronchopulmonary dysplasia (BPD) in infants, chronic obstructive pulmonary disease (COPD)/emphysema1, pulmonary fibrosis2, lung adenocarcinoma3,4, and respiratory infections like Severe Acute Respiratory Syndrome (SARS)5,6 and COVID-197. Deciphering how these crucial alveolar cell types emerge as well as the mechanisms that regulate their fate is imperative for developing therapies that will not only slow or stop disease progression but may even promote lung repair.

As alveologenesis begins, AT1 and AT2 cells arise from a common pool of distal progenitors (DP)4,8,9,10,11, with differentiation detected as early as embryonic day 16.5 (E16.5) in mice, save for one study reporting an earlier timepoint12. Upon differentiation, AT1 and AT2 cells are arranged in a salt and pepper pattern13 along a lumenal surface, which, as alveoli mature, interconnect with lumens of anatomically distinct branches to facilitate gas conduction between alveoli14. After development, mature AT2 cells can act as antigen presenting cells in an immune response15,16 (partly directed interferon signaling17,18), as well as facultative stem cells following injury4,11,19,20,21 (directed by Wnt22 and Notch23 signaling). While both AT2 states are critical after infection for alveolar recovery, a recent study found interferon stimulation suppresses repair of the lung epithelium following infection24,25, suggesting that signals driving these states may oppose one another at the cellular level.

Here we show nascent AT2 cells are first detected molecularly at E15.5 as singletons at stereotyped intermediate regions of distal branches. Following basal extrusion, we observe nascent AT2s can form de novo attachments to nearby but anatomically separate epithelia, a step that is likely critical in stitching together alveolar lumen and that we term “interlumenal junctioning”, followed by sporadically patterned AT2 maturation, distinct from the “proximal-to-distal wave” pattern reported to initiate differentiation of AT1s and AT2s earlier13.

We determine that nascent AT2s retain fate plasticity into the first perinatal week, revising the lineage model to include a “window of fate plasticity” spanning from distal progenitors to the AT2 lineage. From screening transcriptional regulators around this window, we identify a critical negative regulator of AT2 fate plasticity, the bZIP transcription factor C/EBPα, that when lost results in AT2-to-AT1 conversion. Previously, C/EBPα has been reported to play a role in epithelial homeostasis26 and is implicated in fate regulation in multiple tissues, including the blood and liver27. Further, we found that the observed AT2-to-AT1 differentiation was mediated in a non-cell autonomous manner by the Notch regulator DLK1, a cell-surface transmembrane protein28 that has been associated with brain and muscle development, stem cell maintenance, lung cancer, and abnormal tissue repair23,29. Observing a pulsed expression of Dlk1 during alveologenesis, we find Dlk1 and Cebpa are both regulated by the polycomb repressive complex (PRC2), and determine these components together constitute an incoherent feed-forward loop. Upon abrogation of this “pulsed generator” circuit, we observe a disruption of AT1/AT2 differentiation. PRC2 is a well-established transcriptional repressor that has been shown to regulate DLK1 expression in neuronal fate selection30,31,32 and recent evidence suggests it also modulates C/EBPα expression in the context of adipocyte differentiation33.

Following injury in the adult lung C/EBPα must be downregulated in AT2s for conversion to AT1. We identify that the dominant negative member of the C/EBP family, CHOP, is induced in AT2s following injury and its upregulation promotes AT2-to-AT1 differentiation. Finally, we observe Cebpa loss also activates a “defensive” AT2 state that is enriched in antipathogen response genes, likely regulated by interferon signaling, and is mutually exclusive from the AT1-associated DATP state34.

Results

Timing and pattern of AT2 cell emergence

To delineate newly forming AT2 cells from distal progenitors and their mature counterparts, we analyzed scRNAseq data of the distal epithelial lineage from a prior study sampling the embryonic, perinatal, and adult timepoints of mouse lung development35 (Fig. 1a). Clustering analysis of the distal epithelium identified both DP and AT1 populations, as well as stratified AT2s into two groups (Fig. 1b) that are largely separate in time (Fig. 1c). While these AT2 groups positively correlate in gene expression (Pearson coefficient = 0.6, Supplementary Fig. 1a), their distinction as a separate cluster was confirmed by coassignment probability (Supplementary Fig. 1b). Taken together with the temporal pattern, we refer to the earlier cluster as the nascent AT2 (nAT2) state, and the later as the mature (mAT2) AT2 state. From this distinction, we were able to identify hundreds of genes unique to either DP, nAT2, or mAT2 cluster (Fig. 1d, Supplementary Fig. 1c and Supplementary Tables 1, 2), and initially focused on two for protein-level validation: Retnla expressed by nAT2s and Cd74 by mAT2s (Fig. 1e and Supplementary Fig. 1d). Retnla encodes a regulator of inflammation, resistin-like molecule (RELM) α, while CD74 is critical in MHC-II antigen processing. In contrast to commonly used markers that label both DP and AT2 lineage starting around E17.5 (such as SFTPC)4,10, RELMα uniquely labels nAT2s at this stage and can be used to distinguish them from both DPs and AT1s, as can be seen at an E17.5 transition zone where the mAT2 marker CD74 is not highly expressed (Fig. 1f and Supplementary Fig. 1e). Further, flow cytometry confirmed at E18.5 that a substantial proportion of marker-expressing AT2s are RELMα⁺ CD74− (Supplementary Fig. 1g), which shifts to RELMα− CD74+ in adult AT2s (Supplementary Fig. 1h).

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

a scRNAseq UMAP generated from a published timecourse study (GSE119228)35. 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.

Given the distinct labeling of nAT2s by RELMα in the embryonic lung, we used it to identify when and where AT2 cells first emerge during alveologenesis. Careful immunostaining revealed nAT2s emerge as early as E15.5 (Fig. 1g), which steadily increases over time (Fig. 1h). At first emergence, SOX9low/RELMα+ and nAT2s are singletons (Supplementary Fig. 1e, f) with retained columnar morphology (Fig. 1i) positioned at anatomical intermediate zones, located between airway smooth muscle (ASM)-covered distal branch stalks and terminal end buds (Fig. 1g, asterisks). After their initial emergence, nAT2s next arise (~E17.5) within distal stalks following ASM to myofibroblast remodeling as previously reported[36](https://www.nature.com/articles/s41467-025-64224-1#ref-CR36 “Gillich, A. St. et al. Alveoli form directly by budding led by a single epithelial cell. bioRxiv https://doi.org/10.1101/2021.12.25.474174

(2021).“), then finally arise at terminal end buds soon after birth4. These observations support a sequential, three-zone model of AT2 differentiation (Fig. 1j, left and Supplementary Fig. 1d, e) that integrates previously conflicting findings that reported AT2 differentiation first occurring either at terminal end buds12 or solely in a proximal-to-distal pattern4,9.

Nascent AT2s junction with nearby but anatomically distinct epithelium

While imaging RELMα at these timepoints, we also observed nAT2s forming connections to lumens across anatomically separate branches (Fig. 2a), a behavior which increased in frequency over time (Fig. 2b). While it is known that during alveolar maturation AT2 cells are capable of simultaneously integrating into lumens of multiple alveoli14,[36](https://www.nature.com/articles/s41467-025-64224-1#ref-CR36 “Gillich, A. St. et al. Alveoli form directly by budding led by a single epithelial cell. bioRxiv https://doi.org/10.1101/2021.12.25.474174

(2021).“), the process by which it occurs has yet to be described. Careful immunostaining of junctioning nAT2s revealed their negativity for AT1 markers (PDPN, RAGE, and HOPX) and positivity for AT2 markers (SFTPC, LAMP1, and MUC1) that is often higher than non-junctioned AT2s, suggesting they may be further along in differentiation (Supplementary Fig. 2a, b). Using high-resolution imaging and 3D reconstruction, we observed that junctions occurred either directly with the second lumen, or indirectly by attachment to another extruded nAT2 (Fig. 2c, Supplementary Movies 1–3 and Supplementary Fig. 2c).

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 Nkx2.1Cre; 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.

To further investigate the dynamics of nAT2 junctioning, we performed timelapse confocal microscopy on Precision Cut Lung Slices (PCLS) isolated from E17.5 murine lungs wherein the distal lung epithelium was labeled with a membrane-tethered GFP (Tg**Nkx2.1-Cre Rosa26**mTmG) so that nAT2s could be readily visualized (Fig. 2d). We observed both direct and indirect nAT2 junctioning—direct when the interstitial distance was short (Fig. 2e) and indirect when the nAT2 was greater than a cell length away (Fig. 2f, Supplementary Fig. 2d and Supplementary Movie 4). During time-lapse microscopy, we also observed cases wherein nAT2s junctioned only temporarily (Supplementary Fig. 2diii). Finally, we observed that for most, if not all junctioning nAT2s, a significant reduction in length of MUC1 domains (Fig. 2g, h). Taken together, we believe our observations are of an initial “interlumenal junctioning” event critical in the process of integrating alveolar lumenal surfaces together (Fig. 2i, j) initiated upon basal extrusion of nAT2s. Further, we hypothesize the manner of junctioning (direct or indirect) is dependent on interstitial distance, and that defective nAT2 junctioning could result in alveolar simplification similar to what is observed in BPD and COPD.

Nascent and mature AT2 state differ in fate plasticity and BH3 regulation

Following their initial specification, nAT2s undergo maturation in the first week or so after birth, which can be observed by the switch from RELMα to CD74 expression (Fig. 3a, b). As RELMα expression is lost in mAT2s, we observe its concomitant upregulation in the airway epithelium (Supplementary Fig. 3a). In contrast to the initial proximal-to-distal wave pattern that AT1/AT2 fate selection is reported to occur4,13, we observe AT2 maturation does not follow this pattern but rather occurs in a sporadic salt-and-pepper pattern (Fig. 3c, d and Supplementary Fig. 3b), suggesting maturation may be governed by a distinct mechanism.

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)39 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.

Given the molecular distinction between nAT2 and mAT2 states, we next sought to determine whether functional differences also exist. One manner in which time-dependent cell states can vary is in fate plasticity, as has been observed in neutrophils37 in vivo and lung epithelial iPSCs38 in culture. To determine whether and how nAT2s and mAT2s differ in fate plasticity, we analyzed two previously generated scRNAseq datasets that sample timepoints either during12 (Fig. 3e–g and Supplementary Fig. 3c–g) or far beyond39 (Fig. 3h–j) alveolar epithelial differentiation. Analyzing first the distal epithelium prior to (E15.5) and during (E17.5) alveolar epithelial differentiation, we performed standard clustering as well as UMAP visualization and found that distal epithelial cells isolated from these timepoints only rarely co-clustered (Supplementary Fig. 3e), indicating no E15.5 progenitor population had significantly adopted either AT1 or AT2 transcriptional program, a central tenet of the early lineage specification model proposed in a prior study12. A weak correlation between one of the E15.5 progenitor clusters (DE3) and the E17.5 progenitor cluster (0.42) was observed (Supplementary Fig. 3e, yellow box), suggesting a shared transcriptional program.

Single-cell transcriptional dynamics analysis was next analyzed to determine when and how the AT1 and nAT2s clusters segregate. Performing pseudotime (Supplementary Fig. 3f), velocity (Supplementary Fig. 3g), as well as integrated PAGA-Velocity analysis (Fig. 3e) all identified E15.5 clusters as the starting timepoint, as well as identified the AT1 cluster as the predicted endpoint (Supplementary Fig. 3f, g). Further, velocity analysis found a substantial proportion of nAT2s to be on a transcriptional trajectory towards the AT1 cluster, suggesting fate plasticity was retained (Fig. 3e, red arrow). To determine whether a subset of nAT2s displayed a biased trajectory towards AT1 differentiation, we established an AT1 and AT2 score based on well-established marker genes9 and observed a clear stratification within the nAT2 cluster (Fig. 3f). Separating the nAT2 cluster by this ratio into two subgroups (High AT1 or Low AT1 gene expression, >0.5 = High AT1/AT2 score), we compared velocity trajectories and observed a clear separation (Fig. 3g), indicating that a nAT2 subset with a high AT1/AT2 ratio is leaving the nAT2 state and transitioning to AT1.

After observing that embryonic nAT2s possibly retain fate plasticity, we sought to determine how long AT2s retain fate plasticity after birth. Using a previously reported dataset that samples across the lifetime of the distal epithelium of the mouse lung39, we performed similar processing as before to remove duplicates and non-distal epithelial cells before performing UMAP visualization and clustering analysis (Fig. 3h, i). Analyzing more widely over time, we again observed earlier progenitor populations, mature AT1 and AT2 clusters, as well as a cluster transitioning between them as previously reported39. Velocity analysis of this intermediate cluster found the cellular trajectories are oriented away from AT2 and towards AT1 (Fig. 3i, red box) similar to our prior observation. Finally, analysis of the timepoints of cells within the AT2→AT1 transitioning clusters revealed most were from the early perinatal period, suggesting fate plasticity was lost as AT2s mature within the first week after birth (Fig. 3j). To confirm this, we performed alveolosphere differentiation assays following our previously described protocol10 and found that both nAT2s as well as early mAT2s (PN3) could give rise to AT1 cells (Fig. 3k, l), confirming fate plasticity was lost primarily in a time-dependent manner, independent of nAT2 or mAT2 state. Taken together, our analysis indicates that fate plasticity, a property of DPs, is retained in newly formed AT2 cells for a period of 1–2 weeks after birth, revising the model of the alveolar epithelial fate selection model to include a window of plasticity (Fig. 3m).

Another property known to vary from early life to maturity is mitochondrial sensitivity to apoptotic stimuli (apoptotic priming)40, which is reduced during maturation in several organs but can be retained in certain stem cell populations41,42. To determine whether apoptotic sensitivity is retained in nAT2s, BH3 profiling was performed on isolated DPs, nAT2s, as well as adult mAT2s. As expected, DPs and nAT2s exhibited heightened sensitivity to pro-apoptotic BH3 peptides as indicated by higher rates of cytochrome c release, while mAT2s were largely unresponsive (Supplementary Fig. 3h). Taken together, our findings indicate the nAT2 state retains both fate plasticity as well as sensitivity to BH3-regulated apoptosis despite being functionally differentiated.

C/EBPα is required to maintain but not select AT2 fate

Given the loss of fate plasticity as nAT2s mature into mAT2s, we next sought to determine how this shift is regulated. Referencing the scRNAseq data we previously used to distinguish nAT2 and mAT2 clusters (Fig. 1a, b) as well as AT2s sequenced more deeply by the Tabula Muris Consortium43, we screened putative transcriptional and epigenetic regulators from a curated list (1678 genes) and identified 67 genes with ≥2-fold enrichment along the AT2 (Supplementary Table 3) lineage (DP→nAT2→mAT2) compared to AT1 (Fig. 4a). Of these, we then considered only the 32 genes with known embryonic lethal or lung defects when knocked out in mice (Fig. 4a and Supplementary Fig. 4a). These genes can be stratified into four categories based on expression pattern over time: constitutive, DP-restrictive, DP/nAT2-restrictive, and nAT2/mAT2-restrictive (Fig. 4a, lower and Supplementary Fig. 4a). Of these, we focused on genes expressed in the observed window of fate plasticity (Supplementary Fig. 4b), which narrowed the list to four genes, Etv5 and Elf5 (constitutive) as well as, Cebpa and Nupr1 (nAT2/mAT2-Restrictive) (Fig. 4b). While both constitutively expressed genes (Etv5 and Elf5) have been investigated in the lung, findings suggest regulatory roles other than fate plasticity, as ETV5 is required primarily to maintain AT2 survival44 while ELF5 appears to broadly regulate aspects of lung epithelial differentiation45. Further, we excluded study of Nupr1 given its primary role is in death regulation, not differentiation46,47. This left Cebpa as the putative regulator, for which we validated the timing of expression via immunostaining in the embryonic lung (Fig. 4c).

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 Nkx2.1Cre; Rosa26mTmG; Cebpa**wt/fl (left) and Nkx2.1Cre; Rosa26mTmG; 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 LyzMCre; Rosa26mTmG; Cebpa**wt/fl (left) and LyzMCre; Rosa26mTmG; 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.

Previous Cebpa research in the developing mouse lung performed knockouts via a Tet-On approach and concluded it was required for alveolar epithelial differentiation48,49. These findings are conflicting with the expression pattern of Cebpa, which is first expressed after AT2 differentiation, not before (Fig. 4b, c and Supplementary Fig. 6b). Given this discrepancy, as well as a subsequent study that questioned the viability of the prior Tet-On approach in the lung50, we first sought to confirm whether Cebpa was required for alveolar epithelial differentiation via a different method of gene deletion. Using a sparse labeling with efficient recombination approach (Nkx2.1-Cre; Rosa26**mTmG/mTmG; Cebpa**fl/fl) wherein a subset of distal bipotent progenitors is both fluorescently labeled and floxed gene recombined10, we tested whether Cebpa loss indeed resulted in suppressed differentiation. Surprisingly, we observed that both AT2 and AT1 fate selection occurred despite the absence of Cebpa (Fig. 4d and Supplementary Fig. 4c), both at the molecular and morphological level.

As we expected Cebpa to play a role later in development consistent to when it is expressed, we next performed a sparse but efficient deletion experiment wherein Cebpa was deleted in a subset of AT2 cells starting in the early perinatal period (~PN5, LyzMCre; Rosa26**mTmG/mTmG; Cebpa**fl/fl). While lineage-traced control AT2 cells maintain their fate into adulthood, a significant portion of AT2 cells lacking Cebpa converted into AT1 cells (Fig. 4e, f), suggesting an increase in AT2 fate plasticity. Finally, we confirmed the result using an AT2-restrictive tamoxifen-inducible Cre recombinase line (Sftpc**CreER) which again found an increase in conversion to AT1 fate both morphologically and molecularly (Supplementary Fig. 4d). Taken together, we determined that C/EBPα, a bZIP transcription factor expressed following AT2 cell specification is required during the perinatal period to maintain, not select, AT2 fate, suggesting it plays a role in suppressing fate plasticity during the transition from nascent to mature AT2 state.

C/EBPα represses a non-cell autonomous AT1 differentiation program driven by DLK1

To determine more precisely how C/EBPα regulates AT2 fate plasticity, we conducted scRNAseq of the alveolar epithelium from both control (Cebpa**wt/fl) and AT2-floxed (Cebpa**fl/fl) lungs, wherein Cre-mediated deletion was induced in the perinatal period using Sftpc**CreER in combination with the iSure-Cre-tdT (Tg**iSureCre-tdT)51 allele. Retrospective identification of cell types via Louvain clustering using Seurat was able to distinguish control (AT2 – Cebpa**wt/fl) and Cebpa floxed (AT2 – Cebpa**fl/fl) AT2s as well as AT1s (Fig. 5a-i, ii and Supplementary Fig. 5a), a subset of which we confirmed were derived from AT2 cells following Cre induction by expression of the Cre-inducible reporter Tg**iSureCre-tdT (Supplementary Fig. 5b, c), confirming our prior lineage tracing experiments. We observed a substantial down- and upregulation of 117 and 116 genes, respectively, upon loss of Cebpa (Supplementary Fig. 5d). To determine how AT2 and AT1 gene expression overall (Supplementary Fig. 5e) was impacted following Cebpa loss, we first used gene scores to broadly assess changes in either AT2 or AT1 related genes (Supplementary Fig. 5g). While no significant upregulation of AT1 genes was observed (suggesting C/EBPα likely d