Extended Data Fig. 2: Dynamics of cell proliferation and apoptosis in hepatic, ventral pancreato-biliary, and dorsal pancreatic organ rudiments.
From: Quantitative lineage analysis identifies a hepato-pancreato-biliary progenitor niche
a–c, Representative IF images of cryosections of mouse embryos at indicated somite stages stained for pH3 (green) and indicated markers. In a, Ecad (red) marks epithelial cells of the endoderm and ectoderm at 0ss. In b, Ecad (blue)/Prox1 (red) mark the ventral foregut (VFG) at 6ss. In c, Prox1 (left panel) marks liver (LV) cells; Pdx1/Prox1 (left panel) or Pdx1/Sox17 (right panel) mark pancreato-biliary PB cells. In c, left and right panels show consecutive sections of the same embryos. VFG, LV, and PB are outlined by white dotted lines. Hoechst dye was used as nuclear counterstain. NF, neural fold. Scale bars, 100μm. d, Quantification of pH3+ cells in PB buds on cryosections (n = 26 embryos). Dot plot shows the numbers of pH3+ cells as % of the total cell count in the PB bud from E8.5 to E11.0 (9-41ss). Proliferating PB cells were counted on consecutive sections of the same embryos stained either for Prox1/Pdx1/pH3 or Sox17/Pdx1/pH3. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns, not significant. e, Quantification of pH3+ dorsal (DP) and ventral (VP) pancreatic cells on cryosections (n = 28 embryos). Dot plot shows the % fraction of pH3+ cells relative to the total cell number in each respective organ rudiment (% of total cell count) from E9.0 to E11.0 (15-44ss). VP cells within the PB bud were defined as described in Extended Data Fig. 5. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. f, Representative whole-mount IF images of embryos at E9.0-E9.75 (16-26 ss) stained for pH3 (green), Prox1 (red) and Pdx1 (blue). LV and PB buds are outlined by dashed white lines. Scale bar, 100μm. g, Measurement of LV and PB bud volumes at the indicated somite stages (n = 129 embryos). 3D organ volumes were reconstructed by measuring the surface area on individual optical sections of confocal z-series of whole-mount IFs (Fig. 1a). Average surface areas in each embryo were then multiplied by tissue thickness. h, Quantification of pH3+ cell numbers in whole-mount IF images of LV and PB buds (n = 71 embryos) normalized to organ bud volume (mm3). pH3 levels were higher in pancreato-biliary rudiments as compared to liver buds at early stages (E8.5; P = 0.0016), while no significant differences between the two organ rudiments were observed at later stages. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. i, Representative image of 18ss embryo stained for the indicated markers shows BrdU (green) labelled cells following a 4h-labelling period. Prox1 (red) marks LV, Prox1/Pdx1 (blue) marks PB progenitors. LV and PB buds are outlined by a dashed white line. Scale bar, 100μm. j, BrdU incorporation in LV and PB progenitors of E8.5 and E10.5 embryos following the indicated BrdU pulse-chasing periods. Dot plot showing the fraction of BrdU-labelled cells relative to the total cell number in each organ rudiment (% of total cell count). Each dot represents the mean from an individual embryo [E8.5: n(1h) = 4, n(2h) = 6, n(4h) = 8; E10.5: n(0.5h) = 3, n(4h) = 5]. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. k, Graph illustrating cell proliferation rates as shown by the % of BrdU+ cells in E8.5 and E10.5 LV and PB organ domains at different labelling periods (Extended Data Fig. 2j). The cell cycle length (tc) was estimated based on BrdU incorporation using linear regression56,57. LV (E8.5) slope = 3.8%/h; estimated cell cycle length = 26.6h (range with 95% confidence interval: 44.7h-19h). PB (E8.5) slope = 5.8%/h; estimated cell cycle length = 17.3h (range with 95% confidence interval: 29.6h-12.2h). LV (E10.5) slope = 5.5%/h; estimated cell cycle length = 18.2h (range with 95% confidence interval: 21h-16.1h). PB (E10.5) slope = 4.8%/h; estimated cell cycle length = 21h (range with 95% confidence interval: 33h-15.4h). No statistically significant differences in average cell cycle length were found between LV and PB progenitors at E8.5 (P = 0.14) or E10.5 (P = 0.36). Two-tailed linear regression t-test. l, Fraction of pH3+/BrdU+ cells in LV and PB buds as % of the total BrdU+ cell population at the indicated pulse-chasing period [n(0.5h) = 3, n(4h) = 3]. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. m, Fraction of BrdU+ early and late mitotic cells in LV and PB buds as % of the total early or late mitotic cell population at the indicated pulse-chasing period [n(0.5h) = 3, n(4h) = 3]. pH3 IF staining intensity was used to identify early (low, punctate pH3 signal) and late (high pH3 signal) mitotic cells. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. n, Fraction of BrdU+ cells in LV, PB and DP buds of E9.5 embryos following the indicated pulse-chasing period [n(2h) = 2, n(4h) = 5]. Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. o, Fraction of BrdU+ cells in DP, VP and GB of E10.5 embryos following the indicated pulse-chasing period (n = 4). Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. p, q, Immunostaining for apoptotic cell marker cleaved caspase 3 (cCas3) in embryos at the indicated somite stages. Representative IF images of cryosections of E8.0 (5ss) Tg(Prox1-EGFP) embryo (p) stained for cCas3 (blue), Prox1 (red) and GFP (green) to detect VFG cells (Prox1+, GFP+) and E9.5 (22ss) embryo (q) stained for cCas3 (blue), Prox1 (red) and Pdx1 (blue). Insets show higher magnifications of boxed regions in grey scale for the indicated channels. Rare apoptotic cells are found in LV and PB bud (arrowheads) as well as surrounding mesenchyme (arrow). Hoechst, nuclear counterstain. Scale bar, 100μm. r, Quantification of apoptotic (cCas3+) cells in VFG, LV, and PB buds on stained cryosections (n = 19 embryos) as shown in p, q. Dot plot shows the fraction of cCas3+ cells as % of total cell count in the indicated organ domains. No statistically significant differences in apoptosis were found between LV and PB buds at E8.5-E9.0 (12-20ss) or E9.5-E10.0 (21-31ss). Mean ± s.d. Two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons test. ns. Data are representative of 3 or more biologically independent experiments with similar results.
