Dll1 marks early secretory progenitors in gut crypts that can revert to stem cells upon tissue damage

Abstract

Lgr5 stem cells reside at small intestinal crypt bottoms, generating both the enterocyte and secretory lineage. Entry into the latter epithelial lineage requires silencing of Notch signaling. The Notch ligand Dll1 is strongly up-regulated in a small subset of immediate stem cell daughters. Lineage tracing utilizing a novel Dll1^{GFP-ires-CreERT2} knock-in mouse reveals that single Dll1^high cells generate small, short-lived clones of all four secretory cell types. In culture, sorted Dll1^high cells can form long-lived organoids when briefly exposed to Wnt3A. When Dll1 cells are genetically marked prior to tissue damage, significant numbers of stem cell tracing events occur. Lineage specification therefore occurs already in the earliest stem cell daughters through Notch lateral inhibition. Yet, specified secretory progenitors display plasticity and can regain stemness upon tissue damage.

The intestinal epithelium represents a unique model for the study of adult stem cell biology and lineage specification. Not only is it the fastest self-renewing tissue in mammals with a turn-over time of 5 days, it also has a simple, highly repetitive layout. Lgr5^high stem cells are intermingled with Paneth cells at the base of the crypt and feed daughter cells into the Transit Amplifying (TA) compartment that fills the remainder of the crypt (1). TA cells undergo approximately 4–5 rounds of rapid cell division, after which they cross the crypt-villus boundary to terminally differentiate into enterocytes or into one of the secretory cell types, i.e goblet cells, tuft cells and enteroendocrine cells (2-6). The Paneth cells represent a fourth secretory cell type that is unusual in its location at crypt bottoms and its relatively long lifespan of 6-8 weeks (7). Paneth cells serve as niche cells, providing Wnt, Notch and EGF signals to stem cells (8). The first binary fate decision of undifferentiated crypt TA cells involves entry into either the enterocyte or the secretory lineage. A simple molecular circuit downstream of the Notch1/2 receptors controls the absorptive/secretory switch (9). Notch signaling activates Hes1 expression in the receiving cell, typically the crypt stem cell or the undifferentiated TA cells (10-11). HES1 in turn represses expression of the Math1 transcription factor that is crucial for entry into the secretory lineage (12-14). While all TA cells in crypts are capable of converting into secretory cells upon acute blockade of Notch (11,15,16), it is currently not known at what level in the stem cell/TA hierarchy this lineage specification occurs physiologically. Stem cells and the bulk of the TA cells express Notch1 and Notch2 (9), while Delta-like 1 (Dll1) and Delta-like 4 (Dll4) function redundantly as Notch ligands (17).

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

While Paneth cells express Dll1 and Dll4, thus activating Notch receptors on adjacent stem cells, it is unclear which cells in the TA compartment express Notch ligands to activate Notch receptors on TA cells. A Dll1 in situ hybridization probe brightly marked rare cells 1-2 cell positions above the stem cell/Paneth cell zone (Fig. 1A). Much weaker signals were obtained in individual cells higher up in the crypt and on the villus. This pattern was reminiscent of zebra fish DeltaD in secretory cells of the intestinal tract (18). The rare scattered Dll1^high cells in crypts had the morphology of TA cells.

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.

To determine the temporal, hierarchical relationship between the Lgr5 stem cells and these Dll1^high cells, we induced lineage tracing in Lgr5^{GFP-ires-CreERT2} knock-in mice crossed to the Cre reporter R26R^LacZ. At various time points post-tamoxifen induction, we analyzed the expression of the stem cell marker gene Lgr5, the Cre reporter LacZ and Dll1 by triple color mRNA in situ hybridization at single cell resolution (19). We thus noted that 1-2 days after the induction of lineage tracing in stem cells, Dll1^high cells were first observed within the marked clone (Fig. 1C versus 1B). This indicated that –as expected- Dll1^high cells derive from Lgr5⁺ stem cells. More importantly, the time course revealed that Dll1^high cells derive from stem cells within 1-2 cell divisions, given that Lgr5 stem cells divide every 24 hours (20). This was consistent with the physical position of the Dll1^high cell, immediately above the stem cell/Paneth cell zone.

To study Dll1⁺ cells, we integrated a GFP-ires-CreERT2 cassette into the start codon of the Dll1 locus (Suppl. Fig. 1). Heterozygous knock-in mice were healthy and fertile and GFP appeared faithfully expressed as assessed by confocal analysis (Fig. 1D). This analysis showed the presence of rare GFP⁺ cells 1-2 cell cells above the stem cell/Paneth cell zone as well as higher up the crypts and villi (Fig. 1E).

Dll1⁺ cells, obtained by Fluorescence-Activated Cell Sorting (FACS) for GFP and for levels of CD24 expression, were subjected to microarray analysis. This revealed that Dll1^GFP+CD24^high, and Dll1^GFP+CD24^low cells correspond to Paneth cells, and enteroendocrine/goblet cells, respectively (Fig. 1E and Suppl. Fig. 2). The Dll1^GFP+CD24^mid cells expressed markers of multiple secretory lineages, suggesting that these cells represent secretory progenitors. Importantly, Dll1^GFP+CD24^mid cells expressed high levels of Math1 and very low levels of Notch1, Notch2 and Hes1, indicative of an inactive Notch signaling pathway (Suppl. Fig. 2). This set them apart from stem cells and the bulk of TA cells that express high levels of Notch1, Notch2 and Hes1 and low levels of Math1 (9,10,14,16).

Dll1^high precursor cells generate short-lived clones of mixed secretory cell content

We then performed a lineage tracing study by crossing the Dll1^{GFP-ires-CreERT2} allele with the Cre reporter allele R26R^LacZ. Adult mice were subjected to a single tamoxifen pulse and the proximal small intestine was analyzed histologically 12 hrs and, 2, 4, 10 and 122 days later. After 12 hrs, single LacZ⁺ cells occurred mainly around the ‘+5’ position, one cell-diameter removed from the uppermost Paneth cell (Fig. 2A and 2B). We concluded that Cre excision was fortuitously restricted to the cells that express highest Dll1 mRNA levels as assessed by in situ hybridization (Fig. 1B) and not by other GFP-expressing cells (Fig. 1D). On day 2, multiple LacZ⁺ cells occurred at the top of the crypt and the villus base (average 1.4 LacZ⁺ cells per tracing crypt/villus unit; range 1-3 LacZ⁺ cells) (Fig. 2C). On day 4, LacZ⁺ cells mainly occurred on villi (average 3.2 LacZ⁺ cells per tracing crypt/villus unit; range 1-7 LacZ⁺ cells), while occasional LacZ⁺ Paneth cells were first noted (Fig. 2D). On day 10, only LacZ⁺ Paneth cells remained (Fig. 2E). On day 122 post-induction, we occasionally detected ribbons of LacZ⁺ cells (average of 9.8 stem cell derived tracings per duodenum), while we never observed tracing in non-induced mice (not shown).

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.

To determine which cell types became LacZ⁺, Dll1^{GFP-ires-CreERT2}-R26R^LacZ mice were induced by tamoxifen injections on 3 sequential days. Marker analysis, performed 4 days later, revealed LacZ⁺ goblet cells (Fig. 3A), Paneth cells (Fig. 3B), tuft cells (Fig. 3C), and enteroendocrine cells (Fig. 3D). Importantly, we never detected LacZ⁺ enterocytes. We concluded that the Dll1^high cells localized at the ‘+5’ position generate short-lived clones exclusively consisting of secretory lineage 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).

Using the multi-color Cre reporter R26R^confetti for tracing (21), we found that a single Dll1⁺ cell could give rise to mixed secretory clones consisting of 2-6 cells. Examples are given in Fig. 3E and 3F.

Dll1^high precursor cells build long-lived organoids when exposed to Wnt

To examine the potential stemness of Dll1⁺ cells in vitro, we sorted and cultured Dll1^GFP+CD24^high, Dll1^GFP+CD24^mid and Dll1^GFP+CD24^low cells. Under standard crypt culture conditions (EGF, Noggin and R-spondin1 in Matrigel; (22)), none of the Dll1⁺ FACS-sorted subpopulations generated organoid structures (Fig. 4A), in sharp contrast to sorted Lgr5⁺ cells (22). However, addition of Wnt-3A enabled Dll1⁺CD24^mid cells, but not Dll1⁺CD24^high or Dll1⁺CD24^low cells, to give rise to organoid structures. Single Dll1^GFP+/CD24^mid cells derived from Dll1^{GFP-ires-CreERT2}_Lgr5^LacZ small intestinal crypts and cultured in the presence of Wnt3A, showed the induction of Lgr5^LacZ+ stem cells at the bottom of crypt equivalents (Fig. 4B). Moreover, the presence and location of Dll1^GFP+ secretory cells in these organoids mimicked the in vivo situation (Fig. 4C). Taken together, Dll1⁺CD24^mid secretory progenitor cells could regain stemness upon Wnt stimulation in culture.

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.

Dll1^high precursor cells revert to stem cells upon tissue damage

Dll1^high precursor cells generate short-lived secretory clones, but rarely generate the “signature” long-lived ribbons that Lgr5^high stem cells will (1). The intestine has a unique capacity to regenerate upon extensive damage. We wondered if the Dll1^high fated precursor cells would be capable of replacing lost stem cells upon extensive radiation damage. We therefore induced Cre expression with tamoxifen in the Dll1^{GFP-ires-CreERT2}-R26R^LacZ mice, one day prior to sublethal irradiation (6.0 Gy gamma at day 0). This dosage of irradiation causes strong reduction of Lgr5⁺ stem cells and major apoptosis in the intestinal crypt (not shown, and 1). The duodenum was subsequently analyzed by LacZ staining at day 28 post irradiation. At this time point, we normally only observe Dll1⁺ cell-derived LacZ⁺ Paneth cells at crypt bottoms. As demonstrated by sectioning (Fig. 4D), stem cell tracing events shown as ribbons of cells emanated from the crypt bottoms which ran up the side of adjacent villi (average of 96.1 stem cell derived tracings per duodenum per mouse)(Fig. 4E). These contiguous ribbons consisted of secretory as well as absorptive cells (i.e. enterocytes). These data unequivocally demonstrated that Dll1^high precursor cells can revert to stem cells upon tissue damage. In our control, i.e. tamoxifen induction at day 0 without radiation, we observed occasional complete LacZ⁺ ribbons which shows that this phenomenon also occasional takes place in a normal intestine (average 9.8 stem cell derived tracings per duodenum per mouse, resp) (Fig. 4E). In our control, i.e. tamoxifen injection at day -14 followed by radiation on day 0, we observed similar number of tracings (average 8.4 stem cell derived tracings per duodenum per mouse) showing that this is a phenomenon which is not caused by Dll1 induction in the intestinal stem cells (Fig. 4E).

In this study, we have investigated Dll1-expressing crypt cells from the perspective of the stem cell hierarchy of the small intestinal epithelium. The cells expressing highest levels of Dll1 typically reside one cell diameter above the top Paneth cell, a position we refer to as “+5”. Lineage tracing using a CreERT2-expressing Lgr5 allele demonstrates that Dll1^high cells are immediate descendants of Lgr5⁺ stem cells. Mice generated to carry a novel allele of Dll1 (by insertion of a cassette containing GFP and CreERT2 into the first coding exon) allowed lineage tracing of Dll1^high cells, which were thus shown to generate small, short-lived clones that uniquely consist of cell types of the secretory lineage. Moreover, we found that the Dll1⁺ cells can revert to stem cells, in vitro when provided with exogenous Wnt signals, and in vivo upon tissue damage.

The following scenario can be scripted. In the stem cell zone, the Dll1⁺ Dll4⁺ Paneth cells trigger Notch1 and Notch2 on stem cells, thus keeping the stem cells from terminal differentiation into the secretory lineage (9,17). Each day, each of the 15 stem cells divides and consequently 15 daughter cells exit the Paneth/stem cell zone. These daughters pass through the ‘+5’ position, thereby losing direct access to Delta ligands. Stochastically, some of these cells lose Notch expression, and strongly up regulate Dll1 expression, thereby setting their own secretory fate. Simultaneously, such a cell can present Dll1 to 6-8 neighboring TA cells. These TA cells will thus maintain an active Notch pathway and will stay fated towards the enterocyte lineage. Thus, the enterocyte/secretory switch appears to be controlled through Notch by lateral inhibition. Many examples illustrate this classical mechanism, which typically operates to induce opposite cell fates within fields of initially identical cells (23)

In addition, we identified another characteristic of the Dll1⁺ cells. In vitro as well as in vivo, these early “fated” progenitors in the intestinal epithelium can revert to stem cells, presumably by regaining proximity to Paneth cells, the source of Wnt, Notch and EGF signals (8). Although not probed here, it appears highly like that the bulk of the TA cells which are underway to become enterocytes rather than secretory cells, can similarly de-differentiate into stem cells. This process of dedifferentiation, or plasticity, is not uncommon. For instance in the Drosophila gonad, both female and male germ cells can regain stem cell identity after initiation of differentiation (24-25). Thus, stemness in the intestine may be regarded as a cellular ‘state’ determined by location, rather than a cellular ‘fate’ determined by history. This plasticity may have implications for views on the occurrence and role of cancer stem cells, as have been described in intestinal cancer (reviewed in 26,27).