Dll1+ secretory progenitor cells revert to stem cells upon crypt damage

Abstract

Lgr5+ intestinal stem cells generate enterocytes and secretory cells. Secretory lineage commitment requires Notch silencing. The Notch ligand Dll1 is expressed by a subset of immediate stem cell daughters. Lineage tracing in Dll1(GFP-ires-CreERT2) knock-in mice reveals that single Dll1(high) cells generate small, short-lived clones containing all four secretory cell types. Lineage specification thus occurs in immediate stem cell daughters through Notch lateral inhibition. Cultured Dll1(high) cells form long-lived organoids (mini-guts) on brief Wnt3A exposure. When Dll1(high) cells are genetically marked before tissue damage, stem cell tracing events occur. Thus, secretory progenitors exhibit plasticity by regaining stemness on damage.

Fig. 1. Dll1 is expressed by rare undifferentiated TA cells that are early descendants of Lgr5 stem cells

(A) In situ hybridization performed on small intestine, suggesting the presence of rare Dll1 mRNA expressing cells above the Paneth cell zone. (B-C) Three color single molecule FISH (LacZ – TMR (green), Dll1 – cy5 (red), and Lgr5 – Alexa594 (blue)) on the intestine of Lgr5^{GFP-ires-CreERT2}-R26R^LacZ mice 1 day (B) and 2 days (C) after cre induction. On day 1, the Lgr5⁺ cells (arrows), but not the Dll1^high precursor (arrow heads), expressed LacZ, while on day 2 also the Dll1^high precursor expressed LacZ (arrow heads). Rarely did we see Dll1^high precursors being LacZ⁺ on day 1. (D) Confocal GFP imaging of Dll1^{GFP-ires-CreERT2} duodenum visualizes Dll1-GFP expressing cells. (E) Confocal GFP imaging of Dll1^{GFP-ires-CreERT2} duodenum, counterstained for the Paneth cell marker lysozyme. GFP marked cells occur 1-2 cell diameters (arrowheads)(so-called ‘+5 position’), and higher, above the stem cell/Paneth cell zone. (F) Purified villi or crypts are dissociated into single cells, and analyzed for Dll1-GFP and CD24-PE by flow cytometry. The dot plots are gated on viable single cells using negative propidium iodide staining and Pulse width/FSC parameters. Discrete populations in Dll1^GFP+ cells are depicted. Subsequent microarray analysis revealed that Dll1^GFP+/CD24^mid, Dll1^GFP+/CD24^high and Dll1^GFP+/CD24^low cells are Paneth cells, secretory progenitors and goblet/enteroendocrine cells, respectively.

Fig. 2. Lineage tracing of Dll1^{GFP-ires-CreERT2} × R26R^LacZ knock-in intestine

(A) Frequency at which LacZ cells first appeared at 12 hrs post-tamoxifen induction at specific positions relative to the crypt bottom (blue line). Quantitative data on the position of LacZ⁺ cells observed upon tamoxifen induction in Lgr5^{GFP-ires-CreERT2} × R26R^LacZ mice are from (1) (green line). Histological analysis of R26R^LacZ activity in Dll1^{GFP-ires-CreERT2} × R26R^LacZ on 12 hours(B), 2days (C), 4days (D) and 10days (E) post Cre-induction revealed that single marked cells grew into short-lived clones of scattered cells.

Fig. 3. Intestinal Dll1⁺ cells are secretory lineage precursors

Double-labeling of LacZ-stained intestine with PAS demonstrates the presence of goblet cells (A, arrow) and Paneth cells (B, arrow) in induced LacZ⁺ (blue) clones. Double-labeling with synaptophysin demonstrates the presence of enteroendocrine cells (C, arrow), while double-labeling with Dcamkl1 demonstrates the presence of Tuft cells (D, arrows) within the induced LacZ⁺ clones. E/F Two examples of confocal analysis of Dll1^{GFP-ires-CreERT2} × R26R^confetti reporter mice shows that a single Dll1⁺ precursor cell gives rise to a Paneth (black arrow) cell and 2 goblet cells (white arrows) (E) or multiple goblet cells (F).

Fig. 4. Dll1 precursors can convert to intestinal stem cells

(A) Dll1^{GFP-ires-CreERT2} crypts are dissociated into single cells. Single FACS purified cells (Dll1^GFP+/CD24^low, Dll1^GFP+/CD24^mid and Dll1^GFP+/CD24^high cells) are cultured +/− Wnt3A and the frequency of organoid formation is determined 10 days after culture. (B-C). Organoids grown from single Dll1^GFP+/CD24^mid cells derived from Dll1^{GFP-ires-CreERT2}/Lgr5^LacZ small intestinal crypts, cultured in the presence of Wnt3A. The expression of Lgr5^LacZ is analyzed by X-gal staining 14 days after culture. LacZ expression is only seen at the bottom of budding structures. (C) The expression of Dll1^GFP staining of cultured organoids analyzed by confocal imaging. Dll1^GFP expression resembles in vivo presence of Dll1⁺ cells. Staining (D) and quantitative analysis (E) of LacZ activity in the duodenum of the Dll1^{GFP-ires-CreERT2} × R26R^LacZ mice upon cre induction followed by radiation 1 day later (exp. group 3). This analysis reveals the formation of numerous LacZ⁺ stem cell ribbons (exp group 3, n = 96.1). Tamoxifen induction at day 0 without radiation (exp. group 1; n= 9.8) and tamoxifen injection at day −14 followed by radiation on day 0 (exp. group 2, n=8.4), resulted in rare background LacZ⁺ ribbons. The values are given as mean +/− standard error of the mean (s.e.m.), derived from the analyses of 8 different mice for each group.