Evidence of secondary Notch signaling within the rat small intestine.

The small intestine is well known for its nutrient-absorbing enterocytes; yet equally crucial for homeostasis is a diverse set of secretory cells, all presumed to originate from the same intestinal stem cell. Despite their major roles in intestinal function and health, understanding how the full spectrum of secretory cell types arises remains a longstanding challenge, largely due to their comparative rarity. Here, we investigate the specification of a rare population of small intestinal epithelial cells found in rats and humans but not mice: CFTR high expressers (CHEs). Using pseudotime trajectory analysis of single-cell RNA-sequencing data from rat jejunum, we provide evidence that CHEs are specified along the secretory lineage and appear to employ a second wave of Notch-based signaling to distinguish themselves from other secretory cells. We validate the transcription factors directing these cells from crypt progenitors and demonstrate that Notch signaling is necessary to induce CHE fate in vivo and in vitro. Our findings suggest that Notch reactivation along the secretory lineage specifies CHEs, which may help regulate luminal pH and have direct relevance in cystic fibrosis pathophysiology.