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

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. METHODS: We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions. CONCLUSION: Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation

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

Etude du rôle du facteur de transcription Neurogenin3 dans le développement des cellules endocrines gastro-entéro-pancréatiques

Les cellules endocrines gastro-entéro-pancréatiques constituent le plus grand système endocrine de l'organisme en terme de nombre de lignées. A lui seul, l'intestin regroupe pas moins de 15 lignages entéroendocrines constituant, avec les entérocytes, les cellules de Paneth et les cellules à mucus, l'épithélium intestinal adulte. Ces quatre lignées cellulaires dérivent de cellules souches localisées dans les cryptes de l'intestin. Grâce à ces cellules souches, l'épithélium intestinal est renouvelé tout au long de la vie.Mon travail de thèse a consisté en l'étude du rôle du facteur de transcription à bHLH (basique-hélice-boucle-hélice) Neurogenin3, Ngn3, dans le développement des cellules endocrines gastro-entéro-pancréatiques chez la souris. Les précédents travaux ont démontré le rôle de Ngn3 dans la spécification des progéniteurs endocrines pancréatiques. Les souris mutantes pour ce gène ne développent aucune cellule endocrine pancréatique, elles sont diabétiques et meurent peu de temps après la naissance. Plusieurs observations faites dans le pancréas exocrine et sur la digestion suggèrent que la mutation du gène ngn3 pourrait avoir un effet sur le développement des cellules endocrines intestinales. J'ai étudié le rôle de Ngn3 dans l'intestin et dans un second temps dans l'estomac au cours du développement embryonnaire et chez l'adulte démontrant que ce facteur de transcription est nécessaire à la spécification des progéniteurs endocrines dans l'intestin mais aussi dans certains lignages endocrines de l'estomac. Mes travaux ont mis en évidence que l'ensemble des cellules entéroendocrines ainsi que les six lignages endocrines gastriques dérivent des cellules Ngn3. Comme c'est le cas dans le pancréas, aucune cellule endocrine intestinale ne se développe chez les mutants ngn3. En revanche, trois des six lignages endocrines gastriques sont présents chez les mutants suggérant la co-existence d'un mécanisme de développement endocrine Ngn3-indépendant et d'un mécanisme Ngn3-dépendant au sein de l'estomac.Dans un second temps, nous avons voulu savoir si Ngn3 était suffisant pour développer des lignages entéroendocrines. En effet, plusieurs travaux ont mis en évidence le potentiel de Ngn3 à développer des cellules endocrines pancréatiques à partir de tissus ou de lignées cellulaires d'origine pancréatique. J'ai surexprimé ngn3 dans la lignée cellulaire mIC-cl2 et dans des épithéliums intestinaux au stade E14.5. Mes résultats mettent en évidence que Ngn3 est capable d'enclencher des programmes de différenciation endocrine dans ces deux modèles via l'expression de facteurs de transcriptions connus pour leur implication dans la différenciation endocrine, et via l'expression de gènes codant pour des hormones.Au cours de ma thèse, j'ai également participé à une collaboration portant sur la caractérisation des déterminants génétiques, parmi lesquels Ngn3, impliqués dans le développement des cellules productrices de ghrelin dans le pancréas. J'ai mis en évidence que la ghrelin est produite dans le pancréas entre le stade embryonnaire E10.5 et jusqu'à 2 semaines de vie post-natale et que cette hormone peut être produite par les cellules a, mais également par un lignage qui lui est propre, les cellules e. De plus, j'ai démontré que les cellules productrices de ghrelin dérivent de progéniteurs Ngn3 et sont perdues chez les mutants ngn3 comme le sont les quatre autres lignages endocrines pancréatiques. L'analyse de souris mutantes pour des facteurs de transcription importants dans le développement des lignages endocrines pancréatiques a permis de mettre en évidence que les cellules e constituent un cinquième lignage endocrine pancréatique qui se développe selon ses propres déterminants génétiques, le distinguant des cellules a productrices de ghrelin. Ainsi, ces travaux ont permis de démontrer le rôle essentiel de Ngn3 dans la spécification des progéniteurs endocrines gastrique et intestinaux ainsi que dans le développement d'un nouveau lignage endocrine pancréatique: le lignage des cellules e.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Characterization of cell-fate decision landscapes by estimating transcription factor dynamics

Transcriptional regulation is a fundamental process during cell subtype specification. By modulating the rate of gene expression dynamically, transcription factors promote cell diversity and functional specialization. Despite their crucial role in cell fate decisions, no experimental assays allow the estimation of transcription factors’ regulatory activity in a high-throughput manner and at the single-cell resolution. Here, we present FateCompass, a computational method for identifying lineage-specific transcription factors across differentiation. Our pipeline uses single-cell RNA sequencing data to infer differentiation trajectories and transcription factor activities. We combined a probabilistic framework with RNA velocities or a differentiation potential to estimate transition probabilities and perform stochastic simulations. Also, we implemented a linear model of gene regulation to learn transcription factor activities. Taking into account dynamic changes and correlations, we identified lineage-specific regulators. We applied FateCompass to an islet cell formation dataset from the mouse embryo, and we found known and novel potential cell-type dependent drivers. Also, when applied to a differentiation protocol dataset of human embryonic stem cells towards beta-like cells, our approach pinpointed undescribed regulators of an off-target population of intestinal-like cells. Thus, as a framework for identifying lineage-specific transcription factors, FateCompass could have broader implications on hypothesis generation to increase the understanding of the gene regulatory networks driving cell fate choices during differentiation

Lack of TCF2/vHNF1 in mice leads to pancreas agenesis

International audienceHeterozygous mutations in the human POU-homeobox TCF2 ( vHNF1, HNF1 β) gene are associated with maturity-onset diabetes of the young, type 5, and abnormal urogenital tract development. Recently, pancreas atrophies have been reported in several maturity-onset diabetes of the young type 5 patients, suggesting that TCF2 is required not only for adult pancreas function but also for its normal development. Tcf2 -deficient mice die before gastrulation because of defective visceral endoderm formation. To investigate the role of this factor in pancreas development, we rescued this early lethality by tetraploid aggregation. We show that TCF2 has an essential function in the first steps of pancreas development, correlated with its expression domain that demarcates the entire pancreatic buds from the earliest stages. Lack of TCF2 results in pancreas agenesis by embryonic day 13.5. At earlier stages, only a dorsal bud rudiment forms transiently and expresses the transcription factors Ipf1 and Hlxb9 but lacks the key transcription factor involved in the acquisition of a pancreatic fate, Ptf1a , as well as all endocrine precursor cells. Regional specification of the gut also is perturbed in Tcf2 -/- embryos as manifested by ectopic expression of Shh and lack of Ihh and Ipf1 in the posterior stomach and duodenum. Our results highlight the requirement of Tcf2 for ensuring both accurate expression of key regulator molecules in the stomach–duodenal epithelium and proper acquisition of the pancreatic fate. This study provides further insights into early molecular events controlling pancreas development and may contribute to the development of cell-replacement strategies for diabetes

Expression and functional studies of the GDNF family receptor alpha 3 in the pancreas

International audienceThe generation of therapeutic β-cells from human pluripotent stem cells relies on the identification of growth factors that faithfully mimic pancreatic β-cell development in vitro . In this context, the aim of the study was to determine the expression and function of the glial cell line derived neurotrophic factor receptor alpha 3 (GFRα3) and its ligand artemin (Artn) in islet cell development and function. GFRα3 and Artn expression were characterized by in situ hybridization, immunochemistry, and qRT-PCR. We used GFRα3-deficient mice to study GFRα3 function and generated transgenic mice overexpressing Artn in the embryonic pancreas to study Artn function. We found that GFRα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and β-cells, while Artn is found in the pancreatic mesenchyme. Adult β-cells lack GFRα3 but α-cells express the receptor. GFRα3 was also found in parasympathetic and sympathetic intra-islet neurons as well as in glial cells in the embryonic and adult pancreas. The loss of GFRα3 or overexpression of Artn has no impact on Ngn3 and islet cell formation and maintenance in the embryo. Islet organization and innervation as well as glucose homeostasis is normal in GFRα3-deficient mice suggesting functional redundancy

Pak3 promotes cell cycle exit and differentiation of beta-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

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