Figure 2.
Dichotomous expansion of fallopian tube mutant clones
(A) Approach for whole-fallopian-tube 3D imaging. The harvested reproductive tract, including the fallopian tube, was first fixed with 4% paraformaldehyde (PFA) for 24 h (Day0–1), followed by shaking in CUBIC clearing reagent 1 (CUBIC-1) at 37°C,110rpm for three days (Day1–4). The tissues were then washed in PBS for 6 h to remove the CUBIC-1 reagent and shaken in CUBIC clearing reagent 2 (CUBIC-2) until day 6. The whole fallopian tubes were imaged with light-sheet microscopy.
(B) Distinct clonal size among GFP+ mutant clones showed by 3D imaging of cleared whole FT from 5 months old MADM mutant mice (n = 4). The yellow box shows a region with a non-expanded GFP+ clone (arrow), and the magenta box shows a highly expanded GFP+ clone. Scale bar = 50 μm.
(C) Violin plot of the clonal size distribution of mutant clones in four independent FTs from mice at 5 months of age, hinting at the existence of two density peaks. The magenta line indicates the 75 percentiles.
(D) The clonal size does not fit into one group distribution, suggesting the existence of distinct groups. The observed clonal size of mutant clones (green bar) from four mice at 5 months was first fitted into one negative binomial distribution (yellow dots), which predicts the observation of one clone >50 cells. Because the observed number of clones >50 cells is 27, significantly higher than predicted, the one distribution hypothesis is rejected. Fisher Exact Test, p < 10−8.
(E) At 1 and 2 months old, the clonal size dichotomy also exists. FTs from 1-month-old or 2-month-old MADM mutant mice (n = 4 for each) were cleared for 3D imaging. The yellow box shows a region with a non-expanded GFP+ clone (arrow), and the magenta box shows a highly expanded GFP+ clone. Scale bar = 50 μm.
(F) Violin plot of the clonal size distribution of mutant clones at 1 and 2 months. Data were pooled from 4 mice for each indicated age. A total of 129 GFP+ mutant clones from 1-month-old mice and 233 clones from 2-month-old mice were analyzed for plotting.
(G) The portion of outlier large clones at the indicated age. Each dot indicates data from one mouse (n = 4 for each age). Data are represented as mean ± SD. Mann-Whitney U test was used.See also Figures S4–S6.