Fig 2: Markers to identify cells of epithelial origin.Identification of intestinal cells of epithelial origin within the porcine small intestine and colon are shown. Immunostaining for EpCAM, a pan-epithelial transmembrane protein, demonstrated expression in the basolateral membrane of all cells in both the small intestine and colon arranged along the luminal monolayer of the epithelial mucosa. Immunostaining for Villin, a protein associated with the microvillar actin filaments, showed a gradient of expression localized to the apical border of small intestinal and colonic epithelial cells with increasing intensity in cells located closer to the lumen. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.

Fig 3: Derivation and Clustering of Mouse Mammary Epithelial Single-Cell Transcriptomes from Embryonic Development to Adulthood(A) Isolation and sequencing of mammary cells from different developmental stages. Two strategies are shown, Chromium Drop-Seq (10× Genomics) and C1-microfluidic capture (Fluidigm), with differential output (gray box).(B) tSNE plot of single-cell transcriptomes from indicated developmental stages.(C) The plot in (B), overlaid with relative expression levels of mammary lineage markers.(D) NMF clustering of single-cell expression profiles (n = 6,059), shown by the white-to-black correlation scale. Colored bars correspond to developmental context (right; y axis) or NMF group (x axis).(E) Projection of NMF groups identified in (D) onto the tSNE plot from (B).(F) tSNE plots for adult basal (Epcam+, Cd49f+, left panel) and E18 cells (right panel) isolated, processed, and plotted separately with colors corresponding to the NMF grouping in (D).

Fig 4: Deletion of Igf2 in the epiblast or endothelium impairs Lz expansion(A) Left: schematic of Igf2 expression in conceptuses with conditional deletion driven by Meox2Cre. Right: immunostaining for YFP (green) in a representative fetus and placenta paraffin section at E12 of gestation, double transgenic for Meox2Cre and Rosa26flSTOPflYFP10 reporter. YFP expression in the placenta is localized to the Lz and Cp (high magnification, inset). Blue—DAPI stain for nuclei; scale bars: 1 mm (low magnification) and 100 µm (high magnification).(B) Fetal and placental growth kinetics, measured as average wet-weights for each genotype per litter (E12: n = 10 L [n = 41 controls {C} and n = 32 Igf2EpiKO]; E14: n = 25 L [n = 114 C and n = 88 Igf2EpiKO]; E16: n = 37 L [n = 154 C and n = 127 Igf2EpiKO]; E19: n = 37 L [n = 164 C and n = 121 Igf2EpiKO]).(C) Absolute volumes of the placental layers (Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone; Cp, chorionic plate), measured by stereology (n = 6 per group).(D) Absolute volumes of Lz components, measured by stereology (LT, labyrinthine trophoblast; MBSs, maternal blood spaces; FCs, fetal capillaries) (n = 6 per group).(E) Left: schematic representation of Igf2 expression in conceptuses with conditional deletion driven by TekCre. Right: representative confocal microscopy of frozen sections from a fetus and its corresponding placenta, double transgenic for TeKCre and Ai9(RCL-tdT) reporter at E16 of gestation. Scale bars: 2 mm (fetus) and 1 mm (placenta).(F) Fetal and placental growth kinetics (E12: n = 5 L [n = 17 C and n = 16 Igf2ECKO]; E14: n = 8 L [n = 26 C and n = 34 Igf2ECKO]; E16: n = 13 L [n = 60 C and n = 46 Igf2ECKO]; E19: n = 7 L [n = 31 C and n = 27 Igf2ECKO]).(G) Absolute volumes of the placental layers measured by stereology (n = 5–7 per group).(H) Absolute volumes of Lz components, measured by stereology (n = 5–7 per group).(I) Double immunostaining for EPCAM (epithelial cell adhesion molecule) (red) and MCT1 (monocarboxylate transporter 1) (green) in a representative frozen placental section at E12 of gestation. EPCAM expression is observed as clusters of positive cells within the Lz placenta. Blue—DAPI (4',6-diamidino-2-phenylindole) stain for nuclei; scale bars: 500 µm (left panel) and 20 µm (right panel).(J) Analysis of EPCAMhigh-positive cells by flow cytometry. Left panel: example of gating used to identify EPCAMhigh-positive cells (the viability dye 7-aminoactinomycin D [7-AAD] was used to exclude dead cells). Right: quantification of placental EPCAMhigh-positive cells at E12 in conceptuses with conditional Igf2 deletion driven by Meox2Cre (n = 10 C and n = 9 Igf2EpiKO from 2 L) or TekCre (n = 8 C and n = 8 Igf2ECKO from 2 L). For all graphs data are shown as averages; error bars represent SD in (C), (D), (G), (H), and (J) or 95% confidence intervals (95% CI) in (B) and (F); N.S.—statistically not significant; *p < 0.05; **p < 0.01; ***p < 0.001 calculated by a mixed effects model in (B) and (F) (see STAR Methods), two-way ANOVA plus Sidak’s multiple comparisons tests in (D) and (H) or unpaired t tests in (C), (G), and (J). See also Figures S1–S3.

Fig 5: Lgr5-expressing cells are bipotent in the embryo and lineage restricted in the adult. a–c Lgr5KI; R26LSL-Tom were injected with 4OHT at stage E17 and adult. Eight weeks later, single-cell suspensions were prepared and viable (DAPI negative) cells were analyzed for expression of the tdTomato fluorescent reporter, EpCAM, and CD49f. The percentage of cells that were tdTomato positive was measured by designating a threshold above which epithelial cells of R26WT measured zero. d Lgr5KI; R26LSL-Tom pregnant mice were injected with 4OHT at stage E17. Eight weeks later, they were harvested, fixed, sectioned, and stained for Keratin 8 (K8, blue), Keratin 14 (K14, green), and tdTomato (red). tdTomato co-staining with K8-expressing luminal cells and K14-coexpressing myoepithelial cells are indicated by the white and gray arrows, respectively