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. Characterization of EEC diversity in the mouse embryonic and post-natal intestine by single cell transcriptomics To dissect the mechanisms governing EEC differentiation during development, we used the Neurog3eYFP mouse model which allows the tracing and purification of all eYFP+ EECs from Neurog3-expressing enteroendocrine progenitors to various differentiated EEC subtypes (Blot et al., 2023; Mellitzer et al., 2004; Piccand et al., 2019). We used a plate-based scRNA-seq technology (Sorting and Robot assisted Transcriptome sequencing, SORT-seq (Muraro et al., 2016)) to profile eYFP+ EECs isolated from small intestine at embryonic day 15.5 (E15.5); at E18.5; and at postnatal day 12 (P12). We analyzed the transcriptional profile of a total 1138 cells, comprising 384 from E15.5 small intestine, 514 from E18.5, and 240 from P12 retaining 14495 genes. Characterization of EEC diversity in the developing human small intestine (SI) using in vitro avatars, human intestinal organoids (HIOs) and transplanted HIOs (tHIOs). HIOs mimicking the developing SI were generated from human induced pluripotent stem cells (hiPSCs) of the NEUROG3-HA-P2A-Venus line, enabling EECs to be isolated and traced (Schreiber et al., 2021). The EEC differentiation process was analyzed in HIOs and tHIOs, a model known to induce tissue maturation after transplantation of HIOs under the kidney capsule of immunocompetent mice. Venus-positive EECs were sorted from HIOs or tHIOs and sequenced using plate-based scRNA-seq, SORT-seq. For HIOs, 633 cells and 16513 genes were kept; for tHIOs, 556 cells and 17511 genes were kept.