Figure 3.
Inhibition of cell proliferation does not prevent branch elongation or branch point generation. (A) Ex vivo cultured mammary glands with epithelial mGFP expression (K14-Cre;R26R-mTmG), were imaged before and after 2 h treatment with 1 µg/ml of Mitomycin C (MMC) for cell cycle repression or vehicle (control) followed by 22 h of culture in normal media (mGFP = gray). (B) To assess the effects of MMC treatment on cell proliferation, control and treated glands were labeled with EdU for 1 h at the end of the culture period and fixed for staining and confocal imaging (maximum intensity projections of EdU in white and mGFP in green). (C) The number of tips was quantified from wide-field images of the mGFP-labeled epithelium (A) before and after culturing (ncontrol = 21 glands from 10 explants, ntreated = 22 glands from 12 explants; four experiments). The effects of cell cycle repression on branching morphogenesis was studied by wide-field time-lapse imaging of epithelial mGFP expressing explants (93 h of imaging with 3-h intervals). (D and E) In the middle of the time-lapse (45–47 h), explants were treated for 2 h either with vehicle (D) or with MMC (E), followed by imaging in normal media. (F) The length of terminal branches was measured as a function of time and the elongation rate (Δ length/time) between control and treated glands, before and after treatment compared (ncontrol = 73 branches from 13 glands, ntreated = 62 branches from 12 glands; two time-lapse experiments). (G) The rate of elongation between adjacent time points was determined for each branch and the average plotted as a function of time. (H) Branching events were scored in each gland by careful observation of time-lapse videos and the number of events that occurred in control and treated glands before and after treatment compared (ncontrol = 13 glands, ntreated = 12 glands). The branching events could be further identified as bifurcations or side-branching events as shown in D and E. (I) To assess their prevalence, the overall percentages of different types of branching events were calculated by pooling of events from different glands (total number of events indicated in the chart). Data shown in C, F, and H represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in G represents average (line) ± SD (shaded region). Statistical significance was assessed with the Wilcoxon’s rank sum test (C), two-tailed paired t test (F), or Wilcoxon’s rank sum test (H); *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Size bars = 100 µm.

Inhibition of cell proliferation does not prevent branch elongation or branch point generation. (A) Ex vivo cultured mammary glands with epithelial mGFP expression (K14-Cre;R26R-mTmG), were imaged before and after 2 h treatment with 1 µg/ml of Mitomycin C (MMC) for cell cycle repression or vehicle (control) followed by 22 h of culture in normal media (mGFP = gray). (B) To assess the effects of MMC treatment on cell proliferation, control and treated glands were labeled with EdU for 1 h at the end of the culture period and fixed for staining and confocal imaging (maximum intensity projections of EdU in white and mGFP in green). (C) The number of tips was quantified from wide-field images of the mGFP-labeled epithelium (A) before and after culturing (ncontrol = 21 glands from 10 explants, ntreated = 22 glands from 12 explants; four experiments). The effects of cell cycle repression on branching morphogenesis was studied by wide-field time-lapse imaging of epithelial mGFP expressing explants (93 h of imaging with 3-h intervals). (D and E) In the middle of the time-lapse (45–47 h), explants were treated for 2 h either with vehicle (D) or with MMC (E), followed by imaging in normal media. (F) The length of terminal branches was measured as a function of time and the elongation rate (Δ length/time) between control and treated glands, before and after treatment compared (ncontrol = 73 branches from 13 glands, ntreated = 62 branches from 12 glands; two time-lapse experiments). (G) The rate of elongation between adjacent time points was determined for each branch and the average plotted as a function of time. (H) Branching events were scored in each gland by careful observation of time-lapse videos and the number of events that occurred in control and treated glands before and after treatment compared (ncontrol = 13 glands, ntreated = 12 glands). The branching events could be further identified as bifurcations or side-branching events as shown in D and E. (I) To assess their prevalence, the overall percentages of different types of branching events were calculated by pooling of events from different glands (total number of events indicated in the chart). Data shown in C, F, and H represent the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Data shown in G represents average (line) ± SD (shaded region). Statistical significance was assessed with the Wilcoxon’s rank sum test (C), two-tailed paired t test (F), or Wilcoxon’s rank sum test (H); *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Size bars = 100 µm.

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