A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

Abstract

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.

Figure 1. Characterization of diphtheria toxin-mediated crypt base columnar cell (CBC) ablation

(a) EGFP is detected on the membrane of Ki67⁺ proliferating CBCs in saline treated Lgr5^DTR/+ animals. (b) One dose of diphtheria toxin (DT) eliminates all DTR-EGFP-positive cells at 24 hours. (c) DT treatment for up to 10 days prevents reappearance of Lgr5-expressing cells. (d) Lgr5 mRNA is normally present at the bottom of the crypts and (e) is not detected after 24 hours or (f) 10 days of DT treatment. (g-i) Crypt architecture is intact after ablation of Lgr5-expressing CBCs. (j-l) Proliferation above the crypt base is normal after ablation of Lgr5-expressing CBCs. (m-o) Extensive apoptosis is observed at the crypt base 24 hours after DT and tapers off by 10 days, but is still higher than controls. (p-r) Electron microscopy shows that CBCs in controls are characterized by slender nuclei and scant cytoplasm. No CBCs remain at the crypt base after one dose or 10 days of DT treatment. The crypt base is filled with granule-rich Paneth cells. (s) Dosing regimen for study of the recovery of Lgr5-expressing CBCs (t) No CBCs are detected 24 hours after DT. (u) A few Lgr5⁺/Ki67⁺ CBCs (arrow) were detected 48 hours after the last dose of DT. (v) More Lgr5⁺/Ki67⁺ CBCs (arrow) recovered after 96 hours.

Figure 2. Maintenance of normal crypt architecture is not mediated by Lgr5-positive cells that have escaped ablation

(a) 10 day lineage tracing of descendants of Lgr5-expressing stem cells shows a blue ribbon emanating from the base of the crypt in a grafted intestine piece from E15 Lgr5^DTR/CreER embryos. (b-e) Normal proliferation and differentiation of intestinal epithelium after loss of Lgr5 gene function. Lgr5-expressing stem cells can give rise to all four major differentiated cell types. X-GAL-positive cells mark Lgr5-positive stem cell progeny, which overlap with differentiated cell markers for goblet (c), Paneth (d) and endocrine cell (e) lineages. (f) Concurrent TAM and DT treatment kills all Lgr5-expressing cells. No progeny of Lgr5-expressing cells (blue) are detected in the grafted intestine. (g-j) No GFP-positive cells are detected but proliferation and differentiation are normal after DT-mediated ablation of Lgr5-expressing CBCs

Figure 3. Bmi1-expressing stem cells are mobilized to compensate for the loss of Lgr5-expressing CBCs

(a) Rare Bmi1-expressing cells are detected at positions 3 to 6 of the crypt base in the duodenum of Bmi1^GFP/+ reporter mice. (b) Increased Bmi1-expressing cells appear at the crypt base upon ablation of Lgr5-expressing CBCs. (c) Higher magnification showing a Bmi1-expressing cell at position 4 of crypt base in Bmi1^GFP/+ reporter mice. (d) Close up view of a crypt with multiple Bmi1-expressing cells after ablation of Lgr5-expressing cells. (e) Dosing regimen for lineage tracing of Bmi1-expressing cell progeny after ablation of Lgr5-expressing CBCs. (f-g) Whole-mount X-GAL staining of the gastrointestinal tract. Proximal to distal gradient of decreasing progeny production by Bmi1-expressing cells is maintained after ablation of Lgr5-expressing CBCs. (h-i) Close up view of X-GAL positive crypts in duodenum. Most of the labeled crypts have less than five X-GAL positive cells in Bmi1CreER;R26R control animals. Ablation of Lgr5-expressing CBCs stimulates production of progeny by Bmi1-expressing cells. 36% of the crypts in the first 5 cm of duodenum now become fully labeled (marked by arrows).

Figure 4. Bmi1-expressing cells give rise to Lgr5-expressing CBCs under normal and injury conditions

(a-c) Bmi1CreER;R26R;Lgr5^DTR/+ animals were dosed with 5 mg TAM and harvested 24 hours later. β-gal-positive cell (red) derived from Bmi1-expressing cells overlap with Lgr5-expressing CBCs (GFP-positive, green) at position 4 (arrow). A non-overlapping β-gal-positive cell was detected at position 7 in the same crypt (asterisk). (d-f) More β-gal-positive cells (red) display overlapping expression (marked by arrow) with Lgr5-expressing CBCs (GFP⁺, green) at 48 hours. (g-i) At 72 hours, clonal expansion from Bmi1-positive stem cells is now evident by a streak of β-gal positive cells migrating upward (red). β-gal-positive clones at lower crypt positions overlap with Lgr5-expressing CBCs (arrow). (j-k) Distribution of the Bmi1 positive stem cell progeny (β-gal⁺ cells) within the crypt at 24 and 48 hours post TAM induction. Bmi1-expressing cells appear as singles throughout the crypt base between positions 1 to 15 (j). More cells are derived from Bmi1-expressing stem cells at 48 hours (k). A significant portion of βgal⁺ cells also express Lgr5 (GFP⁺, green column), at positions 1 to 6. Overlapping cells (green) peak around positions 3, 4, or 5. (l) Dosing regimen used to study the recovery of Lgr5-expressing CBCs from Bmi1-positive cells. (m-o) Bmi1-positive cells give rise to a fully labeled crypt (red), including newly formed Lgr5-expressing CBCs (GFP positive, arrows).