Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage

Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.