ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human.

Enteroendocrine cells (EECs) survey the gut luminal environment and co-ordinate hormonal, immune and neuronal responses to it. They exhibit well characterised physiological roles ranging from the control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in human gut.\n\nThe small molecule Isoxazole-9 (ISX-9) stimulates neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. We used ISX-9 as a tool to explore EEC specification in mouse and human intestinal organoids. ISX-9 increased the number of neurogenin3 (Ngn3) positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors which play roles in mouse EEC specification. Single cell analysis revealed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased towards serotonin-producing enterochromaffin (EC) cells. This coincided with enrichment of organoids with functional EC cells which was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types.\n\nOur data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.