precursor cells generate short-lived secretory clones, but rarely generate the “signature” long-lived ribbons that Lgr5high
stem cells will (1)
. The intestine has a unique capacity to regenerate upon extensive damage. We wondered if the Dll1high
fated precursor cells would be capable of replacing lost stem cells upon extensive radiation damage. We therefore induced Cre expression with tamoxifen in the Dll1GFP-ires-CreERT2
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+
Paneth cells at crypt bottoms. As demonstrated by sectioning (), 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)(). These contiguous ribbons consisted of secretory as well as absorptive cells (i.e. enterocytes). These data unequivocally demonstrated that Dll1high
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) (). 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 ().
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 Dll1high 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 Dll1high 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+
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).