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.