Fig 2: Dclk1+pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential.(A) Scheme of FACS and 3D culture of Dclk1+ and Dclk1− PDAC cells from DKPF mice. (B) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. (C and D) Representative images of tumor spheroids derived from sorted Dclk1− (C) and Dclk1+ (D) PDAC cells at day 7. (E) Size of spheroids were compared (mean ± SEM; Dclk1+, n = 5; Dclk1−, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (F) The number of spheroids larger than 20 µm were compared (mean ± SEM; Dclk1+, n = 5; Dclk1−, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (G and H) Tumor-forming assay of Dclk1+ or Dclk1− PDAC cells. (G) Macroscopic image of the NOD/SCID mice after subcutaneous transplantation of Dclk1+ or Dclk1− PDAC cells into the flank. A thousand of Dclk1+ PDAC cells developed subcutaneous tumors whereas Dclk1− PDAC cells did not. (H) Dilution series showed 100, 500, or 1000 Dclk1+ PDAC cells developed subcutaneous tumors in 16.7%, 25.0%, and 75.0% of NOD/SCID mice, whereas same or larger numbers of Dclk1− PDAC cells did not. Statistical significance of the differences is indicated as p<0.05, chi-squared test. (I–K) Histological analysis of primary xenografts derived from FACS-sorted Dclk1+ PDAC cells. (I) Hematoxylin and Eosin (H and E) staining. (J) Immunostaining for Krt19. (K) Immunostaining for Dclk1. Scale bar, 200 µm (I), 50 µm (J and K). (L) Increasing curve of subcutaneous tumor (mean ± SEM; Dclk1+, n = 3; Dclk1−, n = 4; n: number of mice). Statistical significance of the differences is indicated as *p<0.05, Student’s t-test. (M) Passaged tumor-forming assay of Dclk1+ or Dclk1− xenograft cells collected by FACS. A thousand of Dclk1+ xenograft cells developed subcutaneous tumors whereas Dclk1− xenograft cells did not. (N–P) Histological analysis of passaged xenografts derived from FACS-sorted Dclk1+ primary xenograft cells. (N) Hematoxylin and Eosin (H and E) staining. (O) Immunostaining for Krt19. (P) Immunostaining for Dclk1. Scale bar, 200 µm (N), 50 µm (O and P).Figure 6—source data 1.Growth of pancreatic ductal adenocarcinoma (PDAC) xenograft.Measured value of increasing curve of subcutaneous tumor derived from Dclk1+ PDACs cells sorted by FACS.

Fig 3: DCM–Polr2b labeling reveals gene activity maps from ISC to enterocyte.a, Overview of experimental procedure; mice are labeled with dox and sacrificed at different timepoints to isolate EPCAM+/SLC2A2+ enterocytes that are subjected to MeD-seq. ISC, TA, enterocyte and ubiquitously expressed genes are expected to display different dynamic behavior in time. b, Genome browser view of chromosome 19 showing WGBS (n = 1), MeD-seq (average of n = 3) and RNA-seq (average of n = 3) read count distribution on untreated and 8-day dox-treated enterocytes. c, Correlation plot of DCM gene body labeling comparing MeD-seq and WGBS. Genes that are significantly detected using MeD-seq are highlighted in green. d, Genome browser view of the average normalized MeD-seq DCM (average of n = 3), WGBS (n = 1), RNA-seq (average of n = 3) and ChIP-seq (H3K36me3, H3K4me3 and H3K27ac) reads in the Smoc2 and Alpi loci at different timepoints after the start of dox treatment. e,f, DCM labeling (fold change in DCM reads relative to total and normalized to t = 1 day) of ISCs (e) and enterocyte-specific genes (f). g, UMAP of jejunum scRNA-seq data showing clusters annotated as specific cell types. h, DCM labeling of all significantly labeled genes (negative (t = 0) samples compared to all days after the start of dox treatment) clustered according to the maximum DCM signal. For each cluster, the capture by scRNA-seq is shown as the number of genes without reads (black), expressed in five cells with fewer than five reads (gray) and, when expressed, the percentage of cells with signal (blue to red), and average expression of clustered genes is plotted in the UMAP shown in g. i, Top 200 normalized fold-enriched GO terms for each set of genes peaking at day 1 and day 8. Circle size represents gene number per GO term, and color bar displays the ratio of gene count between day 1 and day 8. j,k, DCM labeling (j) and validation by immunocytochemistry (k) of SLC43A2 and NUP54 expression (FITC; DNA is DAPI stained; representative image shown from n = 5 (NUP54) or n = 3 (SLC43A2) replicates; scale bars, 50 µm).

Fig 4: DCM labeling and propagation in the small intestine.a, Genome browser view of Alpi locus showing DCM-specific MeD-seq reads before and after 1 day of dox treatment (n = 3). RNA-seq (±dox, average of n = 3), POLR2A, H3K36me3 and H3K27ac ChIP-seq tracks from ENCODE are shown below. b, Scatter plot displaying RNA-seq gene expression level in relation to DCM read count per gene before (gray) and after (green) 10 days of dox induction in epithelium of jejunum. c, Gene meta-analysis showing distribution of DCM reads in the top 25%, 25–75% and bottom 25% expressed genes after 3 days of dox treatment (average plotted with ± s.e.m.). d, Pearson correlation analysis comparing DCM and ChIP-seq read count distribution. e, Experimental setup; mice received dox for 2 days with isolation of H2B-GFPhigh and H2B-GFPlow and EPCAM+/SLC2A2+ cells after a 3-day chase through FACS analysis, followed by DNA isolation and MeD-seq. f, Immunocytochemistry detecting H2B-GFP (FITC) and DNA (DAPI) in jejunum of a mouse 3 days after an IP dox pulse (representative image shown from n = 5 replicates; scale bar, 50 µm) g, Calculated DCM propagation rate in two independent experiments. h, Relative distribution of DCM reads in intergenic, exonic, intronic and CpG island sequences in GFPhigh and GFPlow cell fractions. i, In silico prediction of DCM labeling levels after each cell division. The fold change between the simulated diluted +dox sample and background levels (-dox) are plotted. The percentage above each violin indicates the percentage of genes that can still be detected based on their estimated fold change.

Fig 5: Pulmonary endothelial heterogeneity increases after birth(A) Overview of the experimental design including the four time points (E18.5, P1, P7, P21) corresponding to key stages in late lung development. In separate experiments, pups were maintained in chronic hyperoxia from birth until P7. Lungs were isolated, perfused, and digested and endothelial cells isolated by fluorescence-activated cell sorting (FACS) for the live, CD326−CD45−CD31+ population.(B) Violin plots displaying the number of genes per detected per cell in our dataset compared with recently published reports.(C) UMAP and Leiden clustering of 2931 endothelial cells identifying 9 transcriptionally distinct EC subpopulations present during the lung from the early saccular to late alveolar stages of development.(D) UMAP of EC clusters identifying developmental time point of cell origin with E18.5 (blue), P1 (yellow), P7 (orange), and P21 (red).(F) UMAP plots of genes expressed predominantly before (E) and (F) after birth. For (E) and (F), the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell.(G) Dot plot showing level of expression (purple to yellow) and fraction of each population expressing the particular gene (dot size) for distinguishing genes expressed by each cluster. See also Figure S1.