Figure 3. Wnt promotes Lgr5⁺ cell migration.

a, Normalised migration tracks of single Lgr5⁺ cells isolated from Lgr5-EGFP-ires-creERT2;R26R-confetti organoids in control medium, medium supplemented with Wnt3a, co-culture with Paneth cells (PC), or co-culture PC with Wnt inhibitor (IWP2) in Matrigel (n=150 random tracks of Lgr5⁺ cells from 2 organoid lines, 3 biological replicates). b, Mean square displacement (MSD) calculated as a function of time (n=150 cells, 3 biological replicates). Data, mean +/- SEM. c, Schematic representation of experimental setup for analysing cell movement on decellularised intestinal scaffolds. d, The motility of Lgr5⁺ cells was determined along the crypt-villus axis (motility in Z) and along the lateral axis (motility in XY) (n=150-200 cell tracks from 3 decellularised intestines for each control (vehicle) and LGK974 treated group). Data, mean with 95% confidence interval. e, Percentage of border starting clones present in the crypt centre on day 3 (n=59, 109, 75 clones in 7, 4, 4 mice for SI, LI and SI upon LGK974 treatment respectively). Bars, mean +/- SEM. Significance was determined by a two-sided Mann-Whitney test. f, Quantification of retention within the Lgr5⁺ zone of centre- or border-starting clones in crypts of control (solid lines) and LGK974-treated (dashed lines) mice as followed by IVM (n=75 clones in 4 mice). g, Normalised retention probability in the Lgr5+ zone in LGK974-treated SI (n=75 clones in 4 mice, data re-analysed from¹⁶ predicted by model (solid lines, mean with 95% confidence interval) and experimental data (dots). h, Probability of a clone starting either in niche centre (left) or border (right) to be present in the centre, border or to be lost from the Lgr5⁺ zone over time in SI of LGK974-treated mice, comparing data (left bar) and theory (right bar) (n=75 clones in 4 mice). See Supplementary Note, section 2.2.3 for details. Scale bars: 50μm (c).