Rapid crypt cell remodeling regenerates the intestinal stem cell niche after stem cell loss induced by Notch inhibition
Authors: Bohin Natacha , Keeley Theresa M. , Carulli Alexis J. , Carlson Elizabeth A. , Gao Jie , Aifantis Iannis , Rajala Michael W. , Myers Martin G. , Jones Jennifer C. , Brindley Constance D. , Dempsey Peter J. , Samuelson Linda C. 
Published: June 2021

Notch is a cell‐to‐cell signaling pathway that is required for intestinal stem cell (ISC) maintenance. Past studies in adult mice suggested that ISCs contain Notch receptor, while ligand is expressed in neighboring Paneth cells. Notch function as an integral ISC niche factor was shown by decreased ISC number and reduced intestinal epithelial cell proliferation after 5 days of continuous Notch inhibition. In this study, we sought to characterize the acute cellular response to Notch inhibition in the intestinal crypt. Adult mice were treated with a single dose of the pan‐Notch inhibitor dibenzazepine (DBZ) and epithelial cell populations were measured 12 hours to 7 days later. Measurement of ISC markers Lgr5 and Olfm4 by in situ hybridization suggested stem cell loss within 12 hours after Notch inhibition, with reduced stem cell function demonstrated by diminished lineage tracing in Lgr5‐CreERT2; Rosa‐Tomato and Olfm4‐CreERT2; Rosa‐Tomato mice. Analysis of Paneth cells by lysozyme staining and by marker expression in Defensin‐Cre; Rosa‐Tomato mice also showed loss of this cell population within 12 hours post DBZ. Apoptotic cell death in the crypt base was confirmed by observation of increased numbers of cleaved caspase 3‐stained cells, with peak levels 1‐day post‐DBZ, suggesting that Notch signaling is required to maintain ISC‐Paneth cell dyads. After cell loss, there is rapid regeneration of both ISCs and Paneth cells starting at 3 days post DBZ. A surge of proliferation was observed 3–5 days following the DBZ‐induced damage, evocative of a regenerative response induced by ISC injury. During DBZ‐induced crypt remodeling, mRNA abundance of the Notch ligands Dll1 and Dll4 increased, with a marked expansion of Dll1‐ and Dll4‐expressing cells shown by analysis of Dll1‐mCherry and Dll4‐mCherry reporter mice. Increased Notch signaling during the hyperproliferative phase of the response was demonstrated by immunostaining for the Notch signaling component NICD. Lineage tracing studies showed that only a subset of facultative stem cells are competent to replenish the ISC pool; Dll1‐ CreERT2; Rosa‐Tomato mice were observed to form lineage stripes, while HopX‐CreER; Rosa‐ Tomato mice did not. Our study uncovered a rapid, multi‐cellular remodeling response to Notch blockade. Rapid loss of ISCs and Paneth cells is followed by expansion of cells expressing Notch ligands and a surge in Notch signaling and cellular proliferation, with ISC regeneration from Dll1‐expressing cells. These findings suggest that Notch signaling plays a key role in ISC maintenance as well as in regeneration of the stem cell niche after injury.Support or Funding InformationSupported by NIDDK R01 DK096972.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.