Figure 5.
Differential cell motility creates a retrograde flow of cells during branch elongation, which is restricted upon tip bifurcation. (A) Maximum intensity projection of sparsely labeled tdTomato+ cells (R26R-tdTomato-flox/wt, induced by Krt5-rtTA;TetO-Cre in the presence doxycycline) in the E18.5 mammary epithelium (magenta), together with volume rendering of isolated cells (gray). EpCAM staining (cyan, bottom right) was used for creating an epithelial surface rendering to mask mesenchymal and to highlight epithelial tdTomato expression (magenta, top right). (B) Quantification of tdTomato+ cells sphericity in the tip (at ≤100 µm distance from the landmark point in the tip) and duct (at >100 µm distance from the landmark point in the tip; ntip = 27 cells, nduct = 49 cells, from nine branches). (C) Visualization of Fucci2a cell tracks, color-coded for mean velocity (fire LUT, black→white = low→high), in an elongating mammary epithelial branch from a 3D time-lapse confocal video of an ex vivo cultured mammary explant (K14-Cre;R26R-Fucci2a-flox/wt). (D) Comparison of mean velocity of tracked cells between tip (defined as the region visibly enriched with M/G2/S cells) and duct (ntip = 600 cells, nduct = 1,814 cells, from nine elongating branches, four experiments). (E) Distribution of mean velocities of cell tracks with respect to their distance to the leading edge of the tip, where the trends of M/G2/S and G1/G0 tracks are shown as cyan and magenta lines, respectively (nM/G2/S = 785, nG1/G0 = 1,629, from nine branches, four experiments). (F) Percentages of cell tracks per video in the tip and duct that switch from basal outermost cell layer (≤6 µm from the surface of the gland) to inner epithelial compartment (>6 µm from the surface of the gland) and vice versa (n = 9 branches, four experiments). (G) Tendency of inner cells in the tip and duct to move closer to the epithelial surface (calculated based on cell distance to the epithelial surface) in elongating branches (ntip = 403 cells, nduct = 1,212 cells; from nine branches, four experiments). (H) Percentages of cell tracks per video that start within the tip (M/G2/S-enriched region) and end in the duct and vice versa (n = 9 branches, four experiments). (I) We analyzed if cells at the end of their tracks were closer or further away from the leading edge of the tip than where they started (change in cell distance to the tip; positive values = closer, negative values further) and compared cells that started within the tip and duct (ntip = 600 cells, nduct = 1,814 cells, from nine branches, four experiments). (J) The change in cell-leading edge distance was plotted against the starting position of the tracks with the trends of M/G2/S and G1/G0 tracks shown as cyan and magenta lines, respectively (nM/G2/S = 785 cells, nG1/G0 = 1,629, from nine branches, four experiments). (K) Visualization of Fucci2a cell tracks, color-coded according to mean velocity, before tip bifurcation (left), during tip widening (middle), and after cleft formation (right) from 3D time-lapse confocal videos of ex vivo cultured mammary explants (fire LUT, black→white = low→high). (L) Mean velocity of tracks derived from 10 bifurcating tips from seven experiments, divided in two groups based on whether they were closer to future tips (blue) or future cleft (red) and compared to those in the duct (gray) according to whether they started before bifurcation (ntip = 272, ncleft = 77, nduct = 154), during tip widening (ntip = 688, ncleft = 279, nduct = 1,152) or after the cleft became visible (ntip = 1,098, ncleft = 1,143, nduct = 1,645). (M) We analyzed if cells at the end of their tracks were closer or further away from the leading edge/future cleft than where they started and compared cells that started within the parent tip and duct (boundary defined based on curvature of the neck) before bifurcation (ntip = 346, nduct = 174), during tip widening (ntip = 879, nduct = 1,240) and after the cleft first appeared (ntip = 1,997, nduct = 1,534). Correlation between variables in E and J was assessed with the Pearson coefficient (R) and P value for the linear correlation given. Data shown in D, G, I, L, and M represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in B, F, and H represents average ± SD. Statistical significance was assessed with the two-tailed t test (B–D and G), paired two-tailed t test (F and H), and Wilcoxon’s rank sum test (I, L, and M); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Differential cell motility creates a retrograde flow of cells during branch elongation, which is restricted upon tip bifurcation. (A) Maximum intensity projection of sparsely labeled tdTomato+ cells (R26R-tdTomato-flox/wt, induced by Krt5-rtTA;TetO-Cre in the presence doxycycline) in the E18.5 mammary epithelium (magenta), together with volume rendering of isolated cells (gray). EpCAM staining (cyan, bottom right) was used for creating an epithelial surface rendering to mask mesenchymal and to highlight epithelial tdTomato expression (magenta, top right). (B) Quantification of tdTomato+ cells sphericity in the tip (at ≤100 µm distance from the landmark point in the tip) and duct (at >100 µm distance from the landmark point in the tip; ntip = 27 cells, nduct = 49 cells, from nine branches). (C) Visualization of Fucci2a cell tracks, color-coded for mean velocity (fire LUT, black→white = low→high), in an elongating mammary epithelial branch from a 3D time-lapse confocal video of an ex vivo cultured mammary explant (K14-Cre;R26R-Fucci2a-flox/wt). (D) Comparison of mean velocity of tracked cells between tip (defined as the region visibly enriched with M/G2/S cells) and duct (ntip = 600 cells, nduct = 1,814 cells, from nine elongating branches, four experiments). (E) Distribution of mean velocities of cell tracks with respect to their distance to the leading edge of the tip, where the trends of M/G2/S and G1/G0 tracks are shown as cyan and magenta lines, respectively (nM/G2/S = 785, nG1/G0 = 1,629, from nine branches, four experiments). (F) Percentages of cell tracks per video in the tip and duct that switch from basal outermost cell layer (≤6 µm from the surface of the gland) to inner epithelial compartment (>6 µm from the surface of the gland) and vice versa (n = 9 branches, four experiments). (G) Tendency of inner cells in the tip and duct to move closer to the epithelial surface (calculated based on cell distance to the epithelial surface) in elongating branches (ntip = 403 cells, nduct = 1,212 cells; from nine branches, four experiments). (H) Percentages of cell tracks per video that start within the tip (M/G2/S-enriched region) and end in the duct and vice versa (n = 9 branches, four experiments). (I) We analyzed if cells at the end of their tracks were closer or further away from the leading edge of the tip than where they started (change in cell distance to the tip; positive values = closer, negative values further) and compared cells that started within the tip and duct (ntip = 600 cells, nduct = 1,814 cells, from nine branches, four experiments). (J) The change in cell-leading edge distance was plotted against the starting position of the tracks with the trends of M/G2/S and G1/G0 tracks shown as cyan and magenta lines, respectively (nM/G2/S = 785 cells, nG1/G0 = 1,629, from nine branches, four experiments). (K) Visualization of Fucci2a cell tracks, color-coded according to mean velocity, before tip bifurcation (left), during tip widening (middle), and after cleft formation (right) from 3D time-lapse confocal videos of ex vivo cultured mammary explants (fire LUT, black→white = low→high). (L) Mean velocity of tracks derived from 10 bifurcating tips from seven experiments, divided in two groups based on whether they were closer to future tips (blue) or future cleft (red) and compared to those in the duct (gray) according to whether they started before bifurcation (ntip = 272, ncleft = 77, nduct = 154), during tip widening (ntip = 688, ncleft = 279, nduct = 1,152) or after the cleft became visible (ntip = 1,098, ncleft = 1,143, nduct = 1,645). (M) We analyzed if cells at the end of their tracks were closer or further away from the leading edge/future cleft than where they started and compared cells that started within the parent tip and duct (boundary defined based on curvature of the neck) before bifurcation (ntip = 346, nduct = 174), during tip widening (ntip = 879, nduct = 1,240) and after the cleft first appeared (ntip = 1,997, nduct = 1,534). Correlation between variables in E and J was assessed with the Pearson coefficient (R) and P value for the linear correlation given. Data shown in D, G, I, L, and M represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in B, F, and H represents average ± SD. Statistical significance was assessed with the two-tailed t test (B–D and G), paired two-tailed t test (F and H), and Wilcoxon’s rank sum test (I, L, and M); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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