Figure 2.
Tips proliferate during branch elongation, while branch points become cell cycle repressed upon tip bifurcation. (A) Epithelial surface rendering of a bifurcating tip (top) and a non-bifurcating tip (below) generated based on whole-mount EpCAM staining. (B) Analysis of tip width by branch length in terminal branches of the surface rendered E18.5 mammary gland with bifurcating and non-bifurcating tips (nbifurcating = 18, nnon-bifurcating = 103 tips of seven glands, four embryos). (C) Confocal slice across a bifurcating tip and surrounding mesenchyme with Fucci2a expression (M/G2/S; cyan, G1/G0; magenta) and EpCAM-staining (gray). (D) Quantification of cell cycle status in the basal and inner compartment of bifurcating tips based Fucci2a fluorescent nuclei, comparing the emerging daughter tips to the branch point as delineated in the schematic (n = 26 bifurcating tips, four embryos; the branch point was defined as a triangular region running from the cleft to the necks of the daughter tips, as shown in the schematic). (E) Confocal time-lapse imaging was performed on ex vivo cultured mammary glands with epithelial Fucci2a expression (K14-Cre;R26R-Fucci2a-flox/wt), and tip width was measured as shown in the schematic on top from elongating terminal branches, based on an epithelial surface rendering (n = 9 branches, four experiments). (F) Dimensions of the bifurcating tip were measured as indicated in the schematic from time-lapse videos at different time points before and after the appearance of a cleft (T0; n = 10 bifurcations, seven experiments). (G) Captions of a time-lapse imaging series featuring two elongating branches with epithelial Fucci2a expression (size bar = 50 µm). Note the enrichment of M/G2/S phase nuclei in the tips, which was used as a proxy to define the tip border in the absence of a discernable neck. (H) Captions featuring a bifurcating tip with epithelial Fucci2a expression (yellow arrowhead points to the cleft, size bar = 50 µm). (I) Quantification of cell cycle status (% M/G2/S phase nuclei) from confocal time-lapse imaging videos of elongating branches comparing the tip to the trailing duct with basal and inner compartments analyzed separately (n = 9 branches, four experiments, data was pooled from all time points). (J) The relative cell cycle status between the branch point (defined as the triangular region running from the cleft to the necks of the emerging daughter tips) and the daughter tips (the branch point-to-tip ratio of % M/G2/S nuclei) of time-lapse imaged bifurcations was plotted against time normalized according to the appearance of the cleft (T0; n = 9 bifurcating tips, six experiments). (K) The ratio of % M/G2/S nuclei was compared 6 h before, upon (T0), and 6 h after the appearance of the cleft in different videos (n = 9 bifurcating tips, six experiments). Correlation between variables in B was assessed with the Pearson coefficient (R) and P value for the linear correlation given. Data shown in D, I, and K represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in E, F, and J represent average (line) ± SD (shaded region). Statistical significance was assessed with the Wilcoxon’s signed rank test (D) and paired two-tailed t test (I and K); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Tips proliferate during branch elongation, while branch points become cell cycle repressed upon tip bifurcation. (A) Epithelial surface rendering of a bifurcating tip (top) and a non-bifurcating tip (below) generated based on whole-mount EpCAM staining. (B) Analysis of tip width by branch length in terminal branches of the surface rendered E18.5 mammary gland with bifurcating and non-bifurcating tips (nbifurcating = 18, nnon-bifurcating = 103 tips of seven glands, four embryos). (C) Confocal slice across a bifurcating tip and surrounding mesenchyme with Fucci2a expression (M/G2/S; cyan, G1/G0; magenta) and EpCAM-staining (gray). (D) Quantification of cell cycle status in the basal and inner compartment of bifurcating tips based Fucci2a fluorescent nuclei, comparing the emerging daughter tips to the branch point as delineated in the schematic (n = 26 bifurcating tips, four embryos; the branch point was defined as a triangular region running from the cleft to the necks of the daughter tips, as shown in the schematic). (E) Confocal time-lapse imaging was performed on ex vivo cultured mammary glands with epithelial Fucci2a expression (K14-Cre;R26R-Fucci2a-flox/wt), and tip width was measured as shown in the schematic on top from elongating terminal branches, based on an epithelial surface rendering (n = 9 branches, four experiments). (F) Dimensions of the bifurcating tip were measured as indicated in the schematic from time-lapse videos at different time points before and after the appearance of a cleft (T0; n = 10 bifurcations, seven experiments). (G) Captions of a time-lapse imaging series featuring two elongating branches with epithelial Fucci2a expression (size bar = 50 µm). Note the enrichment of M/G2/S phase nuclei in the tips, which was used as a proxy to define the tip border in the absence of a discernable neck. (H) Captions featuring a bifurcating tip with epithelial Fucci2a expression (yellow arrowhead points to the cleft, size bar = 50 µm). (I) Quantification of cell cycle status (% M/G2/S phase nuclei) from confocal time-lapse imaging videos of elongating branches comparing the tip to the trailing duct with basal and inner compartments analyzed separately (n = 9 branches, four experiments, data was pooled from all time points). (J) The relative cell cycle status between the branch point (defined as the triangular region running from the cleft to the necks of the emerging daughter tips) and the daughter tips (the branch point-to-tip ratio of % M/G2/S nuclei) of time-lapse imaged bifurcations was plotted against time normalized according to the appearance of the cleft (T0; n = 9 bifurcating tips, six experiments). (K) The ratio of % M/G2/S nuclei was compared 6 h before, upon (T0), and 6 h after the appearance of the cleft in different videos (n = 9 bifurcating tips, six experiments). Correlation between variables in B was assessed with the Pearson coefficient (R) and P value for the linear correlation given. Data shown in D, I, and K represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in E, F, and J represent average (line) ± SD (shaded region). Statistical significance was assessed with the Wilcoxon’s signed rank test (D) and paired two-tailed t test (I and K); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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