The role ofthe transcription factor GATA-6 in mouse embryonic development

Different members of the GAT A family of transcription factors have been studied extensively in our lab. The role of GAT A-I in the differentiation of erythroid blood cells and of GATA-3 during T-lymphocyte development are two typical examples. GATA-6 is the most recently characterized member of the family. Based on its expression pattern during mouse embryonic developmentfor a role for GATA-6 in cardiogenesis had been speculated. To investigate what role GATA-6 may play during embryogenesis we used targeted inactivation of the gene in Embryonic Stem (ES) cells (chapter 2). Unexpectedly, homozygote mutant embryos die just after implantation at embryonic day 5.5. Generation of chimeric embryos in which the GATA-6 mutant cell population was confmed in either the embryo or to the extraembryonic tissues revealed that the primary defect in GATA-6 null embryos lies in an extraembryonic cell lineage. Further in vivo and in vitro analysis of the mutant embryos suggested that the affected lineage is the visceral yolk sac endoderm, a derivative of the primitive endoderm. Cardiogenesis could not be directly studied since mutant embryos die well before heart development starts (embryonic day 8.5). However, in chimeric embryos, GATA-6 -/- ES cells give rise to cardiomyocytes in apparently normal hearts, possibly due to redundant functions with the coexpressed GATA-4 and -5. In contrast, GATA-6 is the only member of the family that is expressed in the lung endoderm. Following on a published observation showing no contribution of GATA-6 null ES cells to the lung epithelium, we decided to generate more highly chimeric embryos to analyze the development of the lung, which is a derivative of another endoderm lineage, the definitive endoderm (chapter 3). Surprisingly, we found that lung endoderm can be formed from GATA-6 mutant cells. However, this mutant endoderm has subsequent morphogenetic and differentiation defects. The importance of GATA-6 protein levels during lung development was confirmed by a different approach. The gene was overexpressed in transgenic mice with a pulmonary epithelium specific promoter (chapter 4). High levels of the protein resulted in branching defects and more interestingly in a block oflung endoderm differentiation to distal alveolar epithelium

The transcription factor GATA6 is essential for early extraembryonic development

The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue

The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development

Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. We show that Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. TTF1 expression is unaltered in chimeric lungs whereas SPC and CC10 expression are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells, implicating that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation

ACTiCLOUD: Enabling the Next Generation of Cloud Applications

Despite their proliferation as a dominant computing paradigm, cloud computing systems lack effective mechanisms to manage their vast amounts of resources efficiently. Resources are stranded and fragmented, ultimately limiting cloud systems' applicability to large classes of critical applications that pose non-moderate resource demands. Eliminating current technological barriers of actual fluidity and scalability of cloud resources is essential to strengthen cloud computing's role as a critical cornerstone for the digital economy. ACTiCLOUD proposes a novel cloud architecture that breaks the existing scale-up and share-nothing barriers and enables the holistic management of physical resources both at the local cloud site and at distributed levels. Specifically, it makes advancements in the cloud resource management stacks by extending state-of-the-art hypervisor technology beyond the physical server boundary and localized cloud management system to provide a holistic resource management within a rack, within a site, and across distributed cloud sites. On top of this, ACTiCLOUD will adapt and optimize system libraries and runtimes (e.g., JVM) as well as ACTiCLOUD-native applications, which are extremely demanding, and critical classes of applications that currently face severe difficulties in matching their resource requirements to state-of-the-art cloud offerings

Marked intrafamilial variability of exocrine and endocrine pancreatic phenotypes due to a splice site mutation in GATA6

The objective of this study was to describe the clinical characteristics of syndromic neonatal diabetes in a family with a GATA6 mutation. A girl, currently aged 12 years 3 months, was born with intrauterine growth retardation: weight 1600 g (–4.3 SDS) at term. After birth, foramen ovale and patent ductus arteriosus (PDA) were diagnosed by echocardiography. Diabetes was diagnosed on the 9th day after birth. Exocrine pancreatic insufficiency was clinically diagnosed at about 2 years of age and pancreatic agenesis was revealed later by magnetic resonance imaging. Her father had undergone surgery during infancy for PDA and had developed insulin dependent diabetes at 12 years of age. Ultrasound revealed a thin pancreas with normal length and anatomical structure. He has subclinical exocrine pancreatic insufficiency, low insulin needs and no late complications of diabetes up to the age of 40 years. Sequencing of GATA6 identified a heterozygous splicing mutation, 1136-2A>G, in the girl and her father. Testing of the paternal grandparents showed that the mutation was likely to have arisen de novo in the father. Identification of a GATA6 mutation explains the cardiac anomalies and diabetes in this family. This case highlights the marked intrafamilial variability of both exocrine and endocrine pancreatic phenotypes in patients with GATA6 mutations.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site

Efficient and Directive Generation of Two Distinct Endoderm Lineages from Human ESCs and iPSCs by Differentiation Stage-Specific SOX17 Transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively

Immunohistochemical detection and regulation of α5 nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis

<p>Abstract</p> <p>Background</p> <p>α₅nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α₅nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α₅nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays.</p> <p>Results</p> <p>Steady expression of α₅nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α₅expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α₅nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α₅is regulated in part by FoxA2, with possible cooperation by GATA-6.</p> <p>Conclusions</p> <p>Our data reveal a highly regulated temporal-spatial pattern of α₅nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α₅in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α₅-containing nAChRs.</p

Brain Struct Funct

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives