Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

Abstract

The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5- Confetti mice. We find that Lgr5(+) cells in the upper part of the niche (termed 'border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed 'central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5(+) cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

Figure 1. Intravital lineage tracing of Lgr5⁺ cells

a, Cartoon showing a mouse carrying an abdominal imaging window (AIW) to visualize intestinal Lgr5⁺ CBC cells and their Confetti progeny over multiple imaging sessions. b, Lateral projection of a Z-stack and representative XY-images of a crypt at indicated Z-stack positions. The stem cell niche (Z0-13) is defined by Lgr5-GFP fluorescence. The relative position of CBC cells to the most basal cell (row 0) determines location in the central (row 0 to +2, which translates to Z0-6) or border region (row +3 to +4, which translates to Z7-13) of the stem cell niche. Scale bar, 20 μm. c-f, Intravital lineage tracing of RFP-expressing Lgr5⁺ CBC cells located at the centre (c,d) and border (e,f) region. Grey lines indicate crypts, white lines indicate Confetti clones. (d,f) Graphs show time evolution of spatial organization of Confetti clones starting 3 days post-induction. Clone size is divided in central (light green) and border (dark green) CBC cells. Asterisk indicates clones in which all progeny were lost. Scale bar, 20 μm.

Figure 2. Central CBC cells experience a short-term positional advantage in self-renewal potential

a-c, Clonal evolution of a Confetti cell located at the central or border region starting 3 days post-induction. Graphs show: a, average clone size; b, fraction “surviving” clones that contain at least one marked central (top) or border (lower) cell; and c, average size of surviving clones (clones with at least one marked cell). Different colours indicate different regions in the niche. Points show data and lines show fit to the biophysical model (see Fig 3). Error bars represent s.d. d, Fold increase in clone size over three days from a border or central Confetti⁺ CBC cell. Note that central stem cells have a positional advantage over border stem cells. Error bars represent s.e.m., P <0.001 obtained using a Mann Whitney U test. e, Intravital images of the same crypt at indicated times. Note that the yellow cell is truly expelled from the stem cell niche, since GFP-expression was absent in the TA cell region (see charts at indicated time points). Scale bars, 20 μm.

Figure 3. Biophysical model of intestinal stem cell dynamics

a, From the unfolded crypt caricature (left), we synthesize a quasi-one-dimensional biophysical model of the niche region (right) consisting of two domains: border and centre. To conserve cell number, cell rearrangements following stem cell division displace precisely one cell from the border. To capture the range of lineage data, we include 5 channels of stem cell loss/replacement (1-5) defined in the main text. b, Cumulative size distributions of clones derived from a single cell in the centre (left) or border (right). Clone size is defined in both cases by total number of constituent cells in centre and border. Error bars represent s.e.m. Points represent predictions of the model using the same parameters as that inferred from the average dependences (Supplementary Notes).

Figure 4. Recovery of stem cell compartment following ablation of Lgr5⁺ cells challenges model

a, Targeted ablation of Lgr5⁺ cells in Lgr5^DTR:EGFP mice was induced by injection of diphtheria toxin. Shown are representative images pre- and post-ablation. Scale bars, 20 μm. b, Recovery of Lgr5⁺ CBC cells was monitored only in mice where full depletion was confirmed 24 hours after diphtheria toxin injection. Images taken at 72 hours after depletion show representative crypts containing clonal clusters of different sizes (n = 108 crypts in 3 mice). Scale bars, 20 μm c, For all various clone sizes, measured spatial composition (border versus centre) of Lgr5⁺ CBC cells in clusters (grey) were accurately predicted by the biophysical model (black). Error bars represent s.d.