Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation [screen]
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE229584 | GEO | 2023/10/27
REPOSITORIES: GEO
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Project description:Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI) and colon. EECs regulate various aspects of metabolic activity including insulin levels, satiety, gastrointestinal secretion and motility. The generation of different EEC lineages of the SI is incompletely understood. We now report a TFome-wide CRISPR knockout screen in adult human intestinal organoids to identify dominant transcription factors controlling EEC differentiation. ZNF800 is discovered as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional SCRISPR screens for genes that program cell differentiation.
2023-10-27 | GSE229585 | GEO
Project description:Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI) and colon. EECs regulate various aspects of metabolic activity including insulin levels, satiety, gastrointestinal secretion and motility. The generation of different EEC lineages of the SI is incompletely understood. We now report a TFome-wide CRISPR knockout screen in adult human intestinal organoids to identify dominant transcription factors controlling EEC differentiation. ZNF800 is discovered as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional SCRISPR screens for genes that program cell differentiation.
2023-10-27 | GSE229583 | GEO
Project description:Objective: Enteroendocrine cells (EECs) survey the gut luminal environment and co-ordinate hormonal, immune and neuronal responses to it. They exhibit well characterized 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. 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. Methods: We investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real time quantitative PCR, fluorescent activated cell sorting, immunostaining and single cell RNA sequencing. Results: 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. Conclusion: Our 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.
2020-01-08 | GSE143221 | GEO
Project description:Background: Endocrine cells of the digestive system, including the pancreatic endocrine cells (PECs) clustered in the islets of Langerhans and the enteroendocrine cells (EECs) scattered in the intestinal epithelium, play an important role in metabolism. Although EECs and PECs are located in distinct organs, they share many features and several common genes control their differentiation. In this study, we investigated comprehensively the similarity of EECs and PECs by defining their transcriptomic landscape and comparing the regulatory networks controlled by pax6b, a key player in both EECs and PECs. Results: RNA-sequencing was performed on EECs and PECs isolated from wild-type and pax6b mutant zebrafish. Data mining of wild-type zebrafish EEC data confirmed the expression of orthologs for most known mammalian EEC hormones but also revealed the expression of three additional neuropeptide hormones (Proenkephalin-a, Calcitonin-a and Adcyap1a) not yet reported to be expressed by EECs in any species. Comparison of transcriptomes from EECs, PECs and other zebrafish tissues highlights a very close similarity between EECs and PECs, with more than 70 % of genes being expressed in both endocrine cell types. Comparison of Pax6b-regulated genes in EECs and PECs revealed a significant overlap. pax6b loss-of-function does not affect the total number of EECs and PECs but instead disrupts the balance between cell subtypes, leading to an increase of ghrelin- and motilin-like expressing cells in both the intestine and pancreas at the expense of other endocrine cells such as beta- and delta-cells in the pancreas and pyyb-expressing cells in the intestine. Finally, we show that the homeodomain of Pax6b is dispensable for its action in both EECs and PECs. Conclusion: This study highlights the close relatedness of EECs and PECs at the transcriptomic and regulatory levels, supporting the hypothesis of a common phylogenetic origin and underscoring the potential implication of EECs in metabolic diseases such as Type 2 diabetes.
2020-08-01 | GSE149081 | GEO
Project description:Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity and systemic metabolism and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences with murine EECs, including in hormones, sensory receptors and transcription factors. Notably, several novel hormone-like molecules were identified. Inter-EEC communication is exemplified by Secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
2020-05-15 | PXD017468 | Pride
Project description:Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration statues as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors are not limited to EECs but are also found in other organs, such as the brain (e.g. Cck and Sst) as well as in the pancreas (e.g. Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly-generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice, and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I, but not D and L cells in the apical villi of the small intestine. The targeting of EECs only in villi is likely caused by the intrinsic Vil1 expression, limiting our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here for the investigation of mature EECs.
2024-01-15 | GSE250433 | GEO
Project description:In this study, similarities between EECs and β-cells were evaluated in detail. To obtain specific subtypes of EECs, cell sorting by flow cytometry was conducted from STC-1 cells (a heterogenous EEC line), and each single cell was cultured and passaged. Five EEC subtypes were established according to hormone expression, measured by quantitative RT-PCR and immunostaining: L, K, I, G and S cells expressing glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, cholecystokinin, gastrin and secretin, respectively. Global microarray gene expression profiles revealed a higher similarity between each EEC subtype and MIN6 cells (a β-cell line) than between C2C12 cells (a myoblast cell line) and MIN6 cells, and all EEC subtypes were highly similar to each other. Genes for insulin secretion-related proteins were mostly enriched in EECs.
2018-09-11 | GSE119747 | GEO
Project description:Adjacent stroma, including subepithelial fibroblasts, are believed to coordinate the differentiation process of epithelial cells, but the mechanisms are not well understood. Glial cell line-derived neurotrophic factor (GDNF) is expressed in the intestinal Pdgfra high subepithelial myofibroblasts (SEMFs), while the GDNF receptor RET is expressed in a subset of enteroendocrine cells (EECs), indicating regulatory crosstalk. In this experiment, single RET+ intestinal cells were sorted the gene expression profile was compared to non-RET cells. This provided knowledge about RET+ cell types. RET+ cells were upregulated with most of the EEC markers, indicating that RET+ cells are expressed in EECs. We also observed the differences in the gene expression profile of stromal-derived GDNF ligand on intestinal organoids. Organoids were derived from intestinal crypts of WT mice C57BL/6J and treated with 500ng/ml of Gdnf and Gfra1. GDNF-treated organoids induced the expression of EEC genes such as Pyy, Tac1, Tph1, and Cck, indicating enhanced differentiation of EC cell and L-I-N cell lineages. This highlights a stroma-epithelium crosstalk pathway regulating the differentiation of intestinal EEC subtypes.
2024-10-17 | GSE259326 | GEO
Project description:Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.
2024-01-10 | GSE238276 | GEO
Project description:Diabetes is a major chronic disease with an excessive healthcare burden on society. A coding variant (p.Arg192His) in the transcription factor PAX4 is uniquely and reproducibly associated with altered risk for type 2 diabetes (T2D) in East Asian populations, whilst rare PAX4 alleles have been proposed to cause monogenic diabetes8. In mice, Pax4 is essential for beta cell formation but neither the role of diabetes-associated variants in PAX4 nor PAX4 itself on human beta cell development and/or function are known. Here, we demonstrate that non-diabetic carriers of either the PAX4 p.Arg192His or a newly identified p.Tyr186X allele exhibit decreased pancreatic beta cell function. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content and altered hormone gene expression. Deletion of PAX4 in isogenic human induced pluripotent stem cell (hiPSC)-derived beta-like cells resulted in de-repression of alpha cell gene expression whilst in vitro differentiation of hiPSCs from carriers of PAX4 p.192His and p.186X alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content. In silico and in vitro studies showed that these PAX4 alleles cause either reduced PAX4 expression or function. Correction of the diabetes-associated PAX4 alleles reversed these phenotypic changes. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function and its contribution to type 2 diabetes risk.
2023-08-05 | GSE203264 | GEO