Intestinal crypts remodel in response to deletion of Notch pathway ligands from Paneth cells

INTRODUCTION: Notch signaling is a principal niche pathway crucial for intestinal stem cells (ISCs), directing ISC self-renewal and lineage commitment to absorptive enterocytes. Because Notch signaling requires cell-cell contact, Paneth cells are considered to be the Notch niche cells as they neighbor each ISC and express the key Notch ligands Dll1 and Dll4. However, no functional studies have investigated the Paneth cell-specific role in the Notch niche. Additionally, ISCs function normally after Paneth cell ablation, yet how they maintain Notch signaling without Paneth cells remains unknown. We aimed to define the ISC response to Paneth cell-specific Notch ligand depletion. We hypothesized that loss of DLL1 and DLL4 in Paneth cells inhibits Notch signaling in ISCs, leading to loss of ISCs as well as impaired proliferation and differentiation. METHODS: To deplete Dll1 and Dll4 in Paneth cells, we crossed Dll1 f/f ; Dll4 f/f mice with a constitutive Paneth cell-specific Cre reporter mouse ( Defensin4-Cre; Rosa-LSL-tdTomato ). To deplete these Notch ligands after intestinal maturation, we crossed Dll1 f/f ; Dll4 f/f mice to a tamoxifen-inducible Paneth cell Cre strain ( Lysozyme-CreER T2 ; Rosa-LSL-tdTomato ), and induced ligand gene deletion in adult mice by treatment with 100mg/kg tamoxifen for 5 days, harvesting intestines 2 days later. Crypt cells were characterized by histological analysis of proliferation (EdU), and ISC (OLFM4), Paneth cell (tdTomato, LYZ, MMP7, UEA1), goblet cell (MUC2, UEA1), and endocrine cell (CHGA) marker expression. RESULTS: Surprisingly, constitutive Paneth cell-specific deletion of Dll1 and Dll4 did not affect overall morphology or proliferation of the intestinal epithelium, suggesting that normal ISC function was maintained throughout development. However, analysis of the ISC marker and Notch target OLFM4, showed a loss of ISC Notch signaling at the crypt base. Instead, cells expressing OLFM4 were shifted higher into the transit-amplifying region of the crypt, indicating that ISCs migrated away from defective Paneth cells and next to Notch ligand-expressing progenitors in the upper crypt. Paneth cells remained clustered at the crypt base, exhibiting hyperplasia with increased numbers of marker-expressing cells. Furthermore, the villus epithelium harbored mislocated tdTomato-positive cells that co-expressed Paneth and goblet cell markers, a feature of Paneth cell progenitors. These changes imply a drive to generate new Paneth cells to replace the ones lacking Notch ligands. In addition, increased numbers of endocrine cells were observed at the crypt base; although their numbers were not sufficient to replace the Paneth cell Notch niche. Like the constitutive model, tamoxifen-inducible deletion of Dll1 and Dll4 in Paneth cells promoted OLFM4 mislocation to the upper crypt, indicating ISC movement out of the crypt base. Interestingly, cleaved caspase 3 staining revealed increased apoptotic cells at the crypt base after inducible Dll1 and Dll4 deletion, suggesting that acute cell death plays a role in the crypt cell remodeling. CONCLUSION: Paneth cell-specific Dll1 and Dll4 deletion induces crypt remodeling to rebuild the ISC Notch niche. This project was funded by NIH R01 DK118023 awarded to L.C. Samuelson. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.