The Homeodomain-Containing Transcription Factors Arx and Pax4 Control Enteroendocrine Subtype Specification in Mice

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3+ endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3+) and in early differentiating (ChromograninA−) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4

Genetic regulation of RNA splicing in human pancreatic islets

Background Non-coding genetic variants that influence gene transcription in pancreatic islets play a major role in the susceptibility to type 2 diabetes (T2D), and likely also contribute to type 1 diabetes (T1D) risk. For many loci, however, the mechanisms through which non-coding variants influence diabetes susceptibility are unknown. Results We examine splicing QTLs (sQTLs) in pancreatic islets from 399 human donors and observe that common genetic variation has a widespread influence on the splicing of genes with established roles in islet biology and diabetes. In parallel, we profile expression QTLs (eQTLs) and use transcriptome-wide association as well as genetic co-localization studies to assign islet sQTLs or eQTLs to T2D and T1D susceptibility signals, many of which lack candidate effector genes. This analysis reveals biologically plausible mechanisms, including the association of T2D with an sQTL that creates a nonsense isoform in ERO1B, a regulator of ER-stress and proinsulin biosynthesis. The expanded list of T2D risk effector genes reveals overrepresented pathways, including regulators of G-protein-mediated cAMP production. The analysis of sQTLs also reveals candidate effector genes for T1D susceptibility such as DCLRE1B, a senescence regulator, and lncRNA MEG3. Conclusions These data expose widespread effects of common genetic variants on RNA splicing in pancreatic islets. The results support a role for splicing variation in diabetes susceptibility, and offer a new set of genetic targets with potential therapeutic benefit.This research was supported by Ministerio de Ciencia e Innovación (BFU2014-54284-R, RTI2018-095666-B-I00), Medical Research Council (MR/L02036X/1), a Wellcome Trust Senior Investigator Award (WT101033), European Research Council Advanced Grant (789055), EU Horizon 2020 TDSystems (667191), ESPACE (874710), and Marie Sklodowska-Curie (643062, ZENCODE). S.B.G was supported by a Juan de la Cierva postdoctoral fellowship (MINECO; FJCI-2017-32090). M.C.A was supported by a Boehringer Ingelheim Fonds PhD fellowship. Work in CRG was supported by the CERCA Programme, Generalitat de Catalunya, Centro de Excelencia Severo Ochoa (CEX2020-001049), and support of the Spanish Ministry of Science and Innovation to the EMBL partnership. Work in Imperial College was supported by NIHR Imperial Biomedical Research Centre. M.I. was supported by a European Research Council consolidator award (101002275). D.J.M.C. and J.A.T. were supported by JDRF grants 9-2011-253, 5-SRA-2015-130-A-N, 4- SRA-2017-473-A-N, and Wellcome grants 091157/Z/10/Z and 107212/Z/15/Z, to the Diabetes and Inflammation Laboratory, Oxford, as well as the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and NIHR Oxford Biomedical Research Centre, and Wellcome Trust Core Award grant 203141/Z/16/Z. D.M.J.C analysis with the UK Biobank Resource was conducted under Application 31295. A.L.G. is a Wellcome Senior Fellow in Basic Biomedical Science and was supported by the Wellcome Trust (095101, 200837, 106130, 203141), the NIDDK (U01DK105535 and UM1 DK126185), and the Oxford NIHR Biomedical Research Centre.Peer Reviewed"Article signat per 20 autors/es: Goutham Atla, Silvia Bonàs-Guarch, Mirabai Cuenca-Ardura, Anthony Beucher, Daniel J. M. Crouch, Javier Garcia-Hurtado, Ignasi Moran, the T2DSystems Consortium, Manuel Irimia, Rashmi B. Prasad, Anna L. Gloyn, Lorella Marselli, Mara Suleiman, Thierry Berney, Eelco J. P. de Koning, Julie Kerr-Conte, Francois Pattou, John A. Todd, Lorenzo Piemonti & Jorge Ferrer"Postprint (published version

Long Non-coding RNAs as Local Regulators of Pancreatic Islet Transcription Factor Genes

The transcriptional programs of differentiated cells are tightly regulated by interactions between cell type-specific transcription factors and cis-regulatory elements. Long non-coding RNAs (lncRNAs) have emerged as additional regulators of gene transcription. Current evidence indicates that lncRNAs are a very heterogeneous group of molecules. For example, selected lncRNAs have been shown to regulate gene expression in cis or trans, although in most cases the precise underlying molecular mechanisms is unknown. Recent studies have uncovered a large number of lncRNAs that are selectively expressed in pancreatic islet cells, some of which were shown to regulate β cell transcriptional programs. A subset of such islet lncRNAs appears to control the expression of β cell-specific transcription factor (TF) genes by local cis-regulation. In this review, we discuss current knowledge of molecular mechanisms underlying cis-regulatory lncRNAs and discuss challenges involved in using genetic perturbations to define their function. We then discuss known examples of pancreatic islet lncRNAs that appear to exert cis-regulation of TF genes. We propose that cis-regulatory lncRNAs could represent a molecular target for modulation of diabetes-relevant genes

Dietary Proteins as Determinants of Metabolic and Physiologic Functions of the Gastrointestinal Tract

Dietary proteins elicit a wide range of nutritional and biological functions. Beyond their nutritional role as the source of amino acids for protein synthesis, they are instrumental in the regulation of food intake, glucose and lipid metabolism, blood pressure, bone metabolism and immune function. The interaction of dietary proteins and their products of digestion with the regulatory functions of the gastrointestinal (GI) tract plays a dominant role in determining the physiological properties of proteins. The site of interaction is widespread, from the oral cavity to the colon. The characteristics of proteins that influence their interaction with the GI tract in a source-dependent manner include their physico-chemical properties, their amino acid composition and sequence, their bioactive peptides, their digestion kinetics and also the non-protein bioactive components conjugated with them. Within the GI tract, these products affect several regulatory functions by interacting with receptors releasing hormones, affecting stomach emptying and GI transport and absorption, transmitting neural signals to the brain, and modifying the microflora. This review discusses the interaction of dietary proteins during digestion and absorption with the physiological and metabolic functions of the GI tract, and illustrates the importance of this interaction in the regulation of amino acid, glucose, lipid metabolism, and food intake

Plasticité et reprogrammation des cellules intestinales

Les cellules endocrines pancréatiques et intestinales partagent de nombreuses caractéristiques moléculaires, cellulaires et fonctionnelles.Particulièrement, leur différenciation repose sur des programmes génétiques similaires contrôlés par le facteur de transcription proendocrine Neurog3. Par conséquent, notre hypothèse est que les cellules souches et progénitrices intestinales pourraient être différenciées en cellules pancréatiques productrices d insulines. Afin de tester cette hypothèse nous avons examiné la plasticité des cellules intestinales murines Neurog3+ in vivo et in vitro, ainsi que la possibilité de reprogrammer des cellules intestinales en cellules pancréatiques. Par traçage cellulaire, nous montrons que les progéniteurs intestinaux Neurog3+ sont multipotents mais, de manière surprenante, se différencient majoritairement en cellules à mucus et à un moindre degré en cellules endocrines et cellules de Paneth. De plus, nous démontrons que l environnement pancréatique n est pas suffisant pour promouvoir la différenciation pancréatiques des cellules Neurog3+ intestinales purifiées. Finalement, nous montrons que l infection des cellules intestinales indifférenciées mIC-cl2 par une combinaison d adénovirus codant pour Pdx1, Neurog3 et Mafa, facteurs de transcription clés du développement des îlots pancréatiques, permet l expression du gène de l insuline, mais n est pas suffisant pour généré des cellules beta sécrétrice d hormones. Par conséquent, des études additionnelles seront nécessaire pour déterminer si les cellules intestinales représentent une source potentielle de cellules beta utilisable pour la thérapie du diabète de type 1.Pancreatic and intestinal endocrine cells share many molecular, cellular and functional characteristics. Particularly, their differentiation during embryogenesis relies on similar genetic programs controlled by the proendocrine transcription factor Neurog3. Therefore, our hypothesis is that intestinal stem or progenitor cells can be coaxed to generate pancreatic endocrine cells such as insulin-producing beta cells. To test this hypothesis we explored the plasticity of mouse intestinal Neurog3+ progenitors in vivo and ex vivo and investigated the possibility to program beta cells from intestinal cells. Using a lineage tracing approach, we showed that, in vivo, intestinal Neurog3+ progenitors are multipotent but surprisingly give rise mainly to goblet cells and to a lower extend to enteroendocrine and Paneth cells. Furthermore, we demonstrated that a pancreatic environment was not sufficient to promote an islet cell fate to purified intestinal Ngn3 progenitor cells and divert them from their enteroendocrine destiny. Finally, we showed that the infection of the undifferentiated mIC-cl2 intestinal cell line with a combination of adenoviruses encoding Pdx1, Neurog3 and Mafa, key transcription factors controlling beta cell differentiation, lead to the induction of the insulin gene but was not sufficient to generate hormone producing beta cells. Consequently additional studies are required to further support the relevance of intestinal cells to generate surrogate beta cells for a cell replacement therapy in type 1 diabetes.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Model of enteroendocrine subtype specification during small intestine development: roles of Arx and Pax4.

<p>Gast-, GIP-, Nts-, Sct-, CCK- and GLP1-expressing cells arise from endocrine progenitors expressing Neurog3 then Pax4 and Arx. Upon Arx inactivation these progenitors are reallocated into Sst-expressing cells while the differentiation of Gast-, GIP-, Nts-, Sct-, CCK- and GLP1-expressing-cells is impaired. Sst- and Serotonin (5-HT)-expressing cells are generated from progenitors expressing Neurog3 then Pax4. Inactivation of Pax4 leads to the up-regulation of Arx and the differentiation of these progenitors into GLP1-expressing cells, while the differentiation of Sst-, Serotonin (5-HT)- Gast-, GIP- and Nts-expressing cells is impaired. Key transcription factors controlling intestinal cell destiny are also indicated.</p

Hormone expression in <i>Pax4</i>-deficient intestine.

<p>(A) Real time RT-PCR analyses of various intestinal hormones mRNAs in <i>Pax4</i>-deficient and control small intestine and colon at 2 days <i>postpartum</i> (n = 4). <i>Gip</i>, <i>Nts</i>, <i>Gast</i>, <i>Sct</i> and <i>Tph1</i> mRNA levels decrease significantly in Pax4 mutant small intestine, <i>Glp1</i> and <i>Ghrl</i> expressions increase in both the small intestine and colon. (B) Quantification of GLP1⁺ cells in Pax4^+/+ (n = 3) and Pax4^−/− P1 ileum (n = 3). GLP1-expressing cells are more abundant in Pax4 mutant ileum. Student's T-test *p<0.05, **p<0.01, ***p<0.001.</p

Loss of enteroendocrine cells in mice alters lipid absorption and glucose homeostasis and impairs postnatal survival

At least 10 enteroendocrine cell types have been identified, and the peptide hormones they secrete have diverse functions that include regulation of glucose homeostasis, food intake, and gastric emptying. Mice lacking individual enteroendocrine hormones, their receptors, or combinations of these have shed light on the role of these hormones in the regulation of energy homeostasis. However, because enteroendocrine hormones have partially overlapping functions, these loss-of-function studies produced only minor phenotypes, and none of the enteroendocrine hormones was shown to be essential for life. To examine the effect of loss of all enteroendocrine cells and hormones on energy homeostasis, we generated mice with intestinal-specific ablation of the proendocrine transcription factor neurogenin 3 (referred to herein as Ngn3Δint mice). Ngn3Δint mice were deficient for all enteroendocrine cells and hormones, and died with a high frequency during the first week of life. Mutant mice were growth retarded and had yellowish stool suggestive of steatorrhea. Subsequent analyses revealed that Ngn3Δint mice had impaired lipid absorption, reduced weight gain, and improved glucose homeostasis. Furthermore, intestinal epithelium of the mutant mice showed an enlarged proliferative crypt compartment and accelerated cell turnover but no changes to goblet and Paneth cell numbers. Enterocytes had shorter microvilli, but the expression of the main brush border enzymes was unaffected. Our data help unravel the role of enteroendocrine cells and hormones in lipid absorption and maintenance of the intestinal epithelium

Arx is expressed in enteroendocrine precursors, downstream of Neurog3.

<p>(A) Real time RT-PCR analysis of <i>Neurog3</i>, <i>ChgA</i>, <i>Arx</i> and <i>Pax4</i> expression in different intestinal regions of 8 weeks old wild-type mice (n = 3). (B) Real time RT-PCR analyses of <i>Neurog3</i> and <i>Arx</i> expression in 8–10 weeks old Villin-Cre;Neurog3^f/f (KO) mice and control Villin-Cre;Neurog3^f/+ (Ctr) mice. <i>Arx</i> expression is completely lost in absence of Neurog3 (n = 5). (C–D) Immunofluorescence on sections of wild-type adult duodenum (C,) and jejunum (D). In C, Arx⁺ cells (red arrows) are localized in the crypt and are ChgA-negative (ChgA⁺ cells, green arrows). In D, Partial overlapping expression of Arx and Neurog3 in the adult mouse intestine is illustrated. Yellow, green and red arrows point to double-labeled, single Neurog3⁺ and single Arx⁺ cells, respectively. (E) <i>In situ</i> hybrization and Immunofluorescence on cross sections of wild-type embryonic pancreas (p) and intestine (i). Blue arrows point to cells expressing <i>Arx</i>, <i>Pax4</i> or <i>Neurog3</i> transcripts. Arx and Pax4 expressions are detected 24 h after Neurog3 expression in enteroendocrine precursors. The red arrow points to an Arx expressing cell. p., proximal; d., distal; duo., duodenum; jej., jejunum; ile., ileum; col., colon; SI, small intestine; p, pancreas; I, intestine. Values are means ± SD. Scale bars (C, left panel) 50 µm, (C right panel, D) 10 µm. ND, Not Detected.</p