Unraveling Enteroendocrine Cell lineage dynamics and associated gene regulatory networks during intestinal development

Enteroendocrine cells (EECs) are rare intestinal epithelial cells producing multiple hormones that regulate essential aspects of digestion and energy. EEC subtypes, their hormone repertoire and differentiation mechanisms from intestinal stem cells have been characterized in the adult intestine. However, the mechanisms underlying EEC subtype specification during development are largely unknown. We characterized EEC lineages and their dynamics during development using both genetically modified mouse and human models (i.e. human intestinal organoids, HIOs, derived from induced pluripotent stem cells and transplanted HIOs, tHIOs), which we analyzed by single-cell RNA sequencing using plate-based SORT-seq technology. Our findings demonstrate that in both mice and humans, the majority of EECs are specified during development through similar differentiation trajectories as observed in the adult intestine. This suggests that EEC subtypes specification occurs independently of fully organized crypt-villus structures and stimulation by diet or microbiota. However, the emergence of certain EEC subtypes depends on tissue maturation. Finally, our integrative approach infers lineage-specific regulators dynamically, identifying new candidates controlling EEC differentiation in the developing human gut.