Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1

BACKGROUND: The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood. RESULTS: By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis. CONCLUSION: In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of gene regulatory networks affected across drug-resistant epilepsies

Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis

A computational framework for complex disease stratification from multiple large-scale datasets.

BACKGROUND: Multilevel data integration is becoming a major area of research in systems biology. Within this area, multi-'omics datasets on complex diseases are becoming more readily available and there is a need to set standards and good practices for integrated analysis of biological, clinical and environmental data. We present a framework to plan and generate single and multi-'omics signatures of disease states. METHODS: The framework is divided into four major steps: dataset subsetting, feature filtering, 'omics-based clustering and biomarker identification. RESULTS: We illustrate the usefulness of this framework by identifying potential patient clusters based on integrated multi-'omics signatures in a publicly available ovarian cystadenocarcinoma dataset. The analysis generated a higher number of stable and clinically relevant clusters than previously reported, and enabled the generation of predictive models of patient outcomes. CONCLUSIONS: This framework will help health researchers plan and perform multi-'omics big data analyses to generate hypotheses and make sense of their rich, diverse and ever growing datasets, to enable implementation of translational P4 medicine

Contrôle de la morphogenèse et de la différenciation du pancréas par des signaux intercellulaires

Pancreas development involves differentiation and morphogenesis from a single population of pluripotent precursors that give rise to endocrine, exocrine and ductal cells, which form islets of Langerhans, acini and excretory ducts. The aim of this work was to contribute to the understanding of intercellular mechanisms that control pancreas differentiation and morphogenesis in the embryo. We have set up a culture model of mouse embryo-derived pancreatic explants and have shown that they recapitulate differentiation and morphogenesis. This allowed us to investigate the role of Activin A, of Stromal cell-Derived-Factor-1 (SDF-1) and of ephrins. When pancreatic buds arise from the endoderm, the pancreatic epithelium contains pluripotent cells. We showed, in our explant culture model, that Activin A can induce intestinal differentiation of these cells and that Sonic Hedgehog induction is necessary and sufficient for this process. These results illustrate the plasticity of the embryonic pancreatic cells and the requirement for a fine tuning of Activin A signaling during pancreas differentiation. After budding, the pancreatic epithelium undergoes a morphogenic process. We showed that the latter is controlled by the signaling pathway induced by SDF-1 binding to its CXCR4 receptor. SDF-1 controls morphogenesis without affecting cell proliferation or endocrine/exocrine cell differentiation. Finally, pancreatic epithelial morphogenesis involves, at a later stage, the formation of burgeoning structures that will give rise to exocrine acini. We studied the expression pattern of ephrins and their receptors during pancreas development and we showed that their genes are expressed mainly in these epithelial burgeoning structures. These data establish a genetic and morphologic basis for the functional study of ephrins and their receptors during pancreas morphogenesis. In conclusion, we have shown, by means of a pancreatic explant model, that the Activin A, SDF-1 and ephrin signaling pathways control pancreas morphogenesis and differentiation.Le développement du pancréas implique des processus de différenciation et de morphogenèse à partir d'une population de précurseurs pluripotents, capables de se différencier en cellules endocrines, exocrines ou canalaires, et de former les îlots de Langerhans, les acini et les canaux excréteurs. L'objectif de ce travail était de contribuer à la compréhension des mécanismes intercellulaires qui contrôlent la différenciation et la morphogenèse du pancréas. Nous avons mis au point des cultures d'explants pancréatiques isolés d'embryons de souris et avons montré qu'elles récapitulent la morphogenèse et la différenciation. Ce modèle nous a permis d'investiguer le rôle de l'Activine A, du Stromal cell-Derived-Factor-1 (SDF-1), et des éphrines. Lorsque les bourgeons pancréatiques se forment à partir de l'endoderme, l'épithélium pancréatique est constitué de cellules pluripotentes. Nous avons montré que l'Activine A peut induire une différenciation intestinale de ces cellules et que l'induction de Sonic Hedgehog est nécessaire et suffisante pour ce phénomène. Ces résultats soulignent la plasticité des cellules pancréatiques embryonnaires et la nécessité d'un contrôle strict de la voie de signalisation de l'Activine A dans la différenciation pancréatique. Après la formation des bourgeons, l'épithélium pancréatique entame un processus de morphogenèse. Nous avons montré que ce dernier est contrôlé par la voie de signalisation induite par la liaison de SDF-1 à son récepteur CXCR4. SDF-1 contrôle la morphogenèse sans affecter la prolifération cellulaire ni la différenciation endocrine ou exocrine. Enfin, la morphogenèse épithéliale implique, à un stade ultérieur, la formation de structures bourgeonnantes qui préfigurent les acini exocrines. Notre étude a révélé que les éphrines et leurs récepteurs sont majoritairement exprimés dans ces structures bourgeonnantes. Ces données établissent une base génique et morphologique pour l'étude de la fonction des éphrines et de leurs récepteurs dans la morphogenèse du pancréas. En conclusion, nous avons montré, grâce à un modèle d'explants de pancréas embryonnaire, que les voies de signalisation de l'Activine A, de SDF-1 et des éphrines contrôlent la différenciation et la morphogenèse du pancréas.Thèse de doctorat en sciences biomédicales (biologie moléculaire et cellulaire)(SBIM 3) -- UCL, 200

Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.

The pancreas develops from the endoderm to give rise to ducts, acini and islets of Langerhans. This process involves extracellular signals of the Transforming Growth Factor beta (TGFbeta) family. The aim of this work was to study the effects of activin A, a member of this family, whose potential role in pancreas differentiation is controversial. To this end, we used pancreatic explants from E12.5 mouse embryos. In culture these explants exhibited spontaneous growth, epithelial morphogenesis and endocrine and exocrine differentiation. Exposure to activin A did not affect exocrine or endocrine differentiation. Surprisingly, activin A induced in the explants the appearance of a large contractile structure surrounded by a cylindrical epithelium, a thick basal lamina and a smooth muscle layer. This structure, the formation of which was prevented by follistatin, was typical of an intestinal wall. Consistent with this interpretation, activin A rapidly induced in the explants the mRNAs for fatty acid binding proteins (FABPs), which are markers of the intestine, but not of the pancreas. We also found that induction of the FABPs was preceded by induction of Sonic hedgehog (Shh), a known inducer of intestinal differentiation in the endoderm. Activin B induced neither Shh nor intestinal differentiation. The activin A-mediated intestinal differentiation was blocked by cyclopamine, an inhibitor of Hedgehog signaling, and it was mimicked by Shh. We conclude that activin A does not appear to affect the exocrine or endocrine components of the pancreas, but that it can promote differentiation of pancreatic tissue into intestine via a Shh-dependent mechanism. These findings illustrate the plasticity of differentiation programs in response to extracellular signals in the pancreas and they shed new light on the regulation of pancreas and intestinal development

Temporal dynamics of a CSF1R signaling gene regulatory network involved in epilepsy

Colony Stimulating Factor 1 Receptor (CSF1R) is a potential target for anti-epileptic drugs. However, inhibition of CSF1R is not well tolerated by patients, thereby prompting the need for alternative targets. To develop a framework for identification of such alternatives, we here develop a mathematical model of a pro-inflammatory gene regulatory network (GRN) involved in epilepsy and centered around CSF1R. This GRN comprises validated transcriptional and post-transcriptional regulations involving STAT1, STAT3, NFκB, IL6R, CSF3R, IRF8, PU1, C/EBPα, TNFR1, CSF1 and CSF1R. The model was calibrated on mRNA levels of all GRN components in lipopolysaccharide (LPS)-treated mouse microglial BV-2 cells, and allowed to predict that STAT1 and STAT3 have the strongest impact on the expression of the other GRN components. Microglial BV-2 cells were selected because, the modules from which the GRN was deduced are enriched for microglial marker genes. The function of STAT1 and STAT3 in the GRN was experimentally validated in BV-2 cells. Further, in silico analysis of the GRN dynamics predicted that a pro-inflammatory stimulus can induce irreversible bistability whereby the expression level of GRN components occurs as two distinct states. The irreversibility of the switch may enforce the need for chronic inhibition of the CSF1R GRN in order to achieve therapeutic benefit. The cell-to-cell heterogeneity driven by the bistability may cause variable therapeutic response. In conclusion, our modeling approach uncovered a GRN controlling CSF1R that is predominantly regulated by STAT1 and STAT3. Irreversible inflammation-induced bistability and cell-to-cell heterogeneity of the GRN provide a theoretical foundation to the need for chronic GRN control and the limited potential for disease modification via inhibition of CSF1R

Cloning and embryonic expression pattern of the mouse Onecut transcription factor OC-2

Onecut (OC) transcription factors are evolutionarily conserved proteins with important developmental functions. They contain a bipartite DNA-binding domain composed of a single cut domain associated with a divergent homeodomain. The human genome contains three Onecut paralogues, Hnf6 (also called Oc1), Oc2 and Oc3. We describe here the cloning of mouse (m) OC-2 and its expression pattern in the mouse embryo. The mOc2 gene was localized on chromosome 18. Analysis of the mOC-2 amino acid sequence revealed overall identities of 67% with mHNF-6 and of 56% with mOC-3, and the presence of functional domains delineated earlier in HNF-6. The sequence of the 153 residue-long cut-homeodomain was very conserved, as it was 92% identical to that of mHNF-6 and 89% identical to that of mOC-3. In situ hybridization showed expression of mOc2 in the developing nervous system and gut endoderm. Like Hnf6, Oc2 was expressed in developing liver and pancreas. As many genes that are targeted by Onecut factors are recognized by both OC-2 and HNF-6, this overlap of expression patterns may have functional implications