Abstract
ObjectiveEnteroendocrine cells (EECs) survey the gut luminal environment and coordinate 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 the human gut. The small molecule isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation. MethodsWe investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real-time quantitative polymerase chain reaction (RT-qPCR), fluorescent-activated cell sorting, immunostaining and single-cell RNA sequencing. ResultsISX-9 increased the number of neurogenin3-RFP (Ngn3)-positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors that play roles in mouse EEC specification. Single-cell analysis showed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased toward serotonin-producing enterochromaffin (EC) cells. Further, we observed enrichment of organoids with functional EC cells that 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. ConclusionOur 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 the mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.
Highlights
The intestinal epithelium is a key interface with our external environment
ISX-9 increases the expression of transcription factors associated with enteroendocrine cells (EECs) differentiation ISX-9 increases the expression of neurogenic differentiation 1 (NeuroD1) and induces differentiation of neuronal [27], cardiac [36] and islet endocrine progenitors [28]
Mouse small intestinal organoids exposed to increasing doses of ISX-9 (48-h treatment) had increased expression of transcription factors known to be important for endocrine specification (Figure 1AeF)
Summary
The intestinal epithelium is a key interface with our external environment It renews itself every 4e5 days, and is composed of five terminally differentiated cell types: the absorptive enterocytes and the secretory Paneth, goblet, tuft and enteroendocrine cells (EECs) [1]. Despite representing only 1% of the epithelium, the EECs constitute the largest hormone-producing tissue and have been described as the gut’s sentinels They sample the luminal, circulating and local tissue environments and coordinate an appropriate response from the epithelium and the immune and nervous systems [2]. Whilst there is a large body of evidence describing the functional roles of gut hormones, comparatively little is known about the factors that control EEC differentiation and assign subset identity. Our data demonstrate proof-of-concept that specific EEC populations can be manipulated with a small molecule, highlight the similarities between mouse and human EEC differentiation and provide a tool to study human EC cells in vitro
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