Fate mapping in mouse demonstrates early secretory differentiation directly from Lgr5+ intestinal stem cells

The intestinal epithelium has a remarkably high turnover in homeostasis. It remains unresolved how turnover is orchestrated at the cellular level and how the behaviour of stem and progenitor cells ensures tissue maintenance. To address this, we combined quantitative fate mapping in three complementary mouse models with mathematical modelling and single-cell RNA sequencing. Our integrated approach generated a spatially and temporally defined model of crypt maintenance that is based on two cycling populations: crypt-based columnar (CBC) and transit amplifying (TA) cells. Validation experiments substantiated the predictions from the model revealing TA cells as the major contributor to the absorptive lineage, while balanced CBC cell fate choices controlled the numbers of cells in the secretory lineage. By unravelling these mechanisms, we gain insights into the process of tissue turnover and provide direct evidence to support the notion of CBC cells as the major driver of the intestinal epithelium replenishment. Lgr5-EGFP-IRES- CreERT2;Rosa26-mTmG mice have been labelled with 4-OH-tamoxifen and traced for 48h (n=4) /72h (n=4). Intestinal epithelial cells have been isolated and stained. Sample from each mouse was stained with a new HTO antibody (Mice traced for 48h #1-4, mice traced 72h #5-8). Single cells were sorted into two eppendorf tubes based on their GFP expression. Cells from 48h/72h clones were sorted as GFPhigh cells, while non-recombined cells were sorted as GFPnegative. Two samples after sorting each had a mix of cells from all the mice. Sample 1 contained GFPneg cells from mice #1,2,5,6 and GFPhigh cells from mice #3,4,7,8. Sample 2 contained GFPneg cells from mice #3,4,7,8 and GFPhigh cells from mice #1,2,5,6.