A conserved Notch–Meis1–Pbx cascade specifies secretory progenitors into spatially diverse intestinal best4 ⁺ cells

best4 ⁺ cells are a recently described vertebrate intestinal epithelial cell type. best4 ⁺ cells are altered in inflammatory bowel disease and colorectal cancer, suggesting that stimulation of their homeostatic replenishment may have therapeutic potential. However, the development and function of best4 ⁺ cells remain unclear. Since mice lack best4 ⁺ cells, we established zebrafish as a tractable in vivo model to observe, manipulate, and remove best4 ⁺ cells in an organismal context. We dissected best4 ⁺ cell developmental regulation in vivo from birth to differentiation and specialization, focusing on factors conserved in best4 ⁺ cells across vertebrates. Lineage tracing demonstrated that best4 ⁺ cells arise from secretory progenitors, where Notch/Dll4 signaling mediates a decision between best4 ⁺ and enterochromaffin cells by triggering meis1b expression. Following specification by meis1b , pbx3a spatially diversifies best4 ⁺ cells, which develop regional heterogeneity in gene expression, intracellular pH, and function. In vivo live imaging and removal of best4 ⁺ cells showed that best4 + cells sense luminal pH changes and extend dynamic luminal and stromal projections, but are not required to restore global luminal pH after challenge. Altogether, this study experimentally delineates best4 ⁺ cell developmental regulation and develops a genetic toolkit to examine their function in vivo, both of which will aid investigating how best4 ⁺ cells are altered or can be restored during disease.