Paneth cell-derived Notch ligands are required for intestinal regeneration after injury

Introduction: Notch signaling is essential for maintaining intestinal stem cell (ISC) activity and for directing epithelial cell fate. Paneth cells have been proposed to be niche cells, providing Notch signal to neighboring stem cells through expression of the key Notch ligands DLL1 and DLL4 on their surface. However, functional studies analyzing Paneth cell-specific roles in stem cell support are lacking. Here, we aimed to define the role of Paneth cell-derived Notch ligands in stem cell function. Hypothesis: We hypothesized that loss of Notch ligands in Paneth cells would reduce stem cell function during homeostasis and with repair after injury. Methods: To deplete DLL1 and DLL4 in Paneth cells, we crossed Dll1f/f; Dll4f/f mice with a constitutive Paneth cell-specific Cre driver mouse (Defensin4-Cre; Rosa-LSL-tdTomato). Intestinal morphology and crypt cells were characterized by histological analysis. Organoid-forming efficiency was used to test stem cell function ex vivo. Crypt proliferation after 12 Gy of whole-body irradiation was used to study regeneration after injury. Results: Mice with DLL1 and DLL4 deleted from Paneth cells were viable with apparently normal gross intestinal morphology. Surprisingly, ISCs (identified by expression of OLFM4 and SLC12A2) were lost from the base of the crypt while proliferating progenitor cells in the upper crypt remained unaffected. This suggests that either some ISCs remained in the upper crypt next to Notch ligand-expressing progenitors or that another cell population compensated for their loss. RNA-seq analysis of sorted Paneth cells after Notch ligand deletion revealed an upregulation of factors involved in fetal reversion and the regenerative response, including Tgfb1, Ly6a, Ly6d, Ly6e, Anxa5, Anxa6, Wnt4, Jak3, and Stat1/4, implying that Paneth cells have remodeled in response to the loss of the crypt base ISCs. We then sought to determine whether an insult would impair the ability of the crypt cells to compensate for the loss of Notch ligands from Paneth cells. Ex vivo, knockout mice showed decreased efficiency in forming enteroids after single-cell dissociation, suggesting an epithelial-specific decline in stem cell function. Likewise, after radiation injury, control mice displayed expanded crypts with an increase in proliferation, while knockout mice possessed reduced numbers of regenerative crypts with fewer proliferating cells. In control mice, the number of ISCs expressing the Notch target OLFM4 also increased during regeneration, whereas, in knockout mice, cells expressing OLFM4 were rare, indicating that Notch signaling after injury was significantly impaired. Conclusion: Thus, Paneth cell-specific Notch ligands DLL1 and DLL4 are necessary for crypt regeneration after injury. This project was funded by R01DK118023, R01DK126451 (LCS), and F32DK142253 (MQ). This abstract was presented at the American Physiology Summit 2026 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.