Blot, F. | bioRxiv Science

bioRxiv2025

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, although EECs need to be functional from birth, as their absence causes severe intestinal malabsorption in newborns, the mechanisms underlying EEC subtype specification during development are largely unknown. We used mouse embryos, human pluripotent stem cell derived intestinal organoid models and single-cell transcriptomics to characterize EEC lineages and dynamics during development. 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. HIGHLIGHTSO_LIMost enteroendocrine cells (EECs) are specified during development. C_LIO_LICrypt-villus organization, diet or microbiota are not essential for EEC formation. C_LIO_LIIn pluripotent stem cell derived human intestinal organoids (HIOs), EECs differentiate along 3 main trajectories, one driven by maturation processes. C_LIO_LINovel Gene Regulatory Networks are identified governing human EEC differentiation in HIOs. C_LI

developmental biology↗

bioRxiv2019

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production

ObjectiveEnteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function, however, the role of Rfx6 in EECs differentiation remained to be elucidated.\n\nMethodsWe examined the molecular, cellular and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes.\n\nResultsRfx6 is expressed in the gut endoderm; later it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, whose differentiation programs are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1- or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine.\n\nConclusionThese studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

developmental biology↗