The salt-and-pepper pattern in mouse blastocysts is compatible with signaling beyond the nearest neighbors.

Embryos develop in a concerted sequence of spatiotemporal arrangements of cells. In the preimplantation mouse embryo, the distribution of the cells in the inner cell mass evolves from a salt-and-pepper pattern to spatial segregation of two distinct cell types. The exact properties of the salt-and-pepper pattern have not been analyzed so far. We investigate the spatiotemporal distribution of NANOG- and GATA6-expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single-cell-based neighborhood analyses. A combination of spatial statistics and agent-based modeling reveals that the cell fate distribution follows a local clustering pattern. Using ordinary differential equations modeling, we show that this pattern can be established by a distance-based signaling mechanism enabling cells to integrate information from the whole inner cell mass into their cell fate decision. Our work highlights the importance of longer-range signaling to ensure coordinated decisions in groups of cells to successfully build embryos.

Maternal age, obesity and hyperglycaemia are associated with a delay in preimplantation embryo development in mouse.

In brief: Fertility has decreased due to advanced maternal age and the rising prevalence of the metabolic syndrome. Using quantitative image analysis methods, we show that these factors are associated with delayed preimplantation embryo development in a mouse model. Abstract: Delayed maternal age, obesity and diabetes are associated with reduced fertility. We investigated how age and obesity/metabolic syndrome impact fertility and hypothesized that its decrease is due to defects in preimplantation embryo development. Three groups of female C57Bl6 mice (12 weeks, 9 months and 1 year old) were fed either a high-fat diet for 8 weeks, to induce obesity and the metabolic syndrome, or a control chow diet. Body weight and composition, glucose tolerance and insulin resistance were assessed. Fecundity was evaluated by mating and pregnancy rates, as well as by the number of embryos. Embryo quality was assessed morphologically, and cell fate composition was analysed in preimplantation embryos by state-of-the-art single-cell quantitative confocal image analysis. The high-fat diet was associated with increased adiposity, glucose intolerance and insulin resistance, especially in the older mice. Fecundity was affected by age more than by the diet. Both age and high-fat diet were associated with reduced cell fate allocation, indicating a delay in the preimplantation embryo development, and with increased expression of GATA3, an inhibitor of placentation. These results support that age and the metabolic syndrome reduce fertility through mechanisms which are present at conception or very early in pregnancy.

Exploring the link between Parkinson's disease and type 2 diabetes mellitus in Drosophila.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Diabetes mellitus (DM) is a metabolic disease characterized by high levels of glucose in blood. Recent epidemiological studies have highlighted the link between both diseases; it is even considered that DM might be a risk factor for PD. To further investigate the likely relation of these diseases, we have used a Drosophila PD model based on inactivation of the DJ-1ß gene (ortholog of human DJ-1), and diet-induced Drosophila and mouse type 2 DM (T2DM) models, together with human neuron-like cells. T2DM models were obtained by feeding flies with a high sugar-containing medium, and mice with a high fat diet. Our results showed that both fly models exhibit common phenotypes such as alterations in carbohydrate homeostasis, mitochondrial dysfunction or motor defects, among others. In addition, we demonstrated that T2DM might be a risk factor of developing PD since our diet-induced fly and mouse T2DM models present DA neuron dysfunction, a hallmark of PD. We also confirmed that neurodegeneration is caused by increased glucose levels, which has detrimental effects in human neuron-like cells by triggering apoptosis and leading to cell death. Besides, the observed phenotypes were exacerbated in DJ-1ß mutants cultured in the high sugar medium, indicating that DJ-1 might have a role in carbohydrate homeostasis. Finally, we have confirmed that metformin, an antidiabetic drug, is a potential candidate for PD treatment and that it could prevent PD onset in T2DM model flies. This result supports antidiabetic compounds as promising PD therapeutics.

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

In Vivo and In Vitro Models of Diabetes: A Focus on Pregnancy.

Diabetes in pregnancy is associated with an increased risk of poor outcomes, both for the mother and her offspring. Although clinical and epidemiological studies are invaluable to assess these outcomes and the effectiveness of potential treatments, there are certain ethical and practical limitations to what can be assessed in human studies.Thus, both in vivo and in vitro models can aid us in the understanding of the mechanisms behind these complications and, in the long run, towards their prevention and treatment. This review summarizes the existing animal and cell models used to mimic diabetes, with a specific focus on the intrauterine environment. Summary of this review.

The transition from local to global patterns governs the differentiation of mouse blastocysts.

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.

Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering.

During mammalian preimplantation, cells of the inner cell mass (ICM) adopt either an embryonic or an extraembryonic fate. This process is tightly regulated in space and time and has been studied previously in mouse embryos and embryonic stem cell models. Current research suggests that cell fates are arranged in a salt-and-pepper pattern of random cell positioning or a spatially alternating pattern. However, the details of the three-dimensional patterns of cell fate specification have not been investigated in the embryo nor in in vitro systems. We developed ICM organoids as a, to our knowledge, novel three-dimensional in vitro stem cell system to model mechanisms of fate decisions that occur in the ICM. ICM organoids show similarities to the in vivo system that arise regardless of the differences in geometry and total cell number. Inspecting ICM organoids and mouse embryos, we describe a so far unknown local clustering of cells with identical fates in both systems. These findings are based on the three-dimensional quantitative analysis of spatiotemporal patterns of NANOG and GATA6 expression in combination with computational rule-based modeling. The pattern identified by our analysis is distinct from the current view of a salt-and-pepper pattern. Our investigation of the spatial distributions both in vivo and in vitro dissects the contributions of the different parts of the embryo to cell fate specifications. In perspective, our combination of quantitative in vivo and in vitro analyses can be extended to other mammalian organisms and thus creates a powerful approach to study embryogenesis.

Structure-activity relationships of 2-arylquinazolin-4-ones as highly selective and potent inhibitors of the tankyrases.

Tankyrases (TNKSs), members of the PARP (Poly(ADP-ribose)polymerases) superfamily of enzymes, have gained interest as therapeutic drug targets, especially as they are involved in the regulation of Wnt signalling. A series of 2-arylquinazolin-4-ones with varying substituents at the 8-position was synthesised. An 8-methyl group (compared to 8-H, 8-OMe, 8-OH), together with a 4'-hydrophobic or electron-withdrawing group, provided the most potency and selectivity towards TNKSs. Co-crystal structures of selected compounds with TNKS-2 revealed that the protein around the 8-position is more hydrophobic in TNKS-2 compared to PARP-1/2, rationalising the selectivity. The NAD(+)-binding site contains a hydrophobic cavity which accommodates the 2-aryl group; in TNKS-2, this has a tunnel to the exterior but the cavity is closed in PARP-1. 8-Methyl-2-(4-trifluoromethylphenyl)quinazolin-4-one was identified as a potent and selective inhibitor of TNKSs and Wnt signalling. This compound and analogues could serve as molecular probes to study proliferative signalling and for development of inhibitors of TNKSs as drugs.

Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells.

Wnt/ß-catenin signalling is a widespread cell signalling pathway with multiple roles during vertebrate development. In mouse embryonic stem (mES) cells, there is a dual role for ß-catenin: it promotes differentiation when activated as part of the Wnt/ß-catenin signalling pathway, and promotes stable pluripotency independently of signalling. Although mES cells resemble the preimplantation epiblast progenitors, the first requirement for Wnt/ß-catenin signalling during mouse development has been reported at implantation [1,2]. The relationship between ß-catenin and pluripotency and that of mES cells with epiblast progenitors suggests that ß-catenin might have a functional role during preimplantation development. Here we summarize the expression and function of Wnt/ß-catenin signalling elements during the early stages of mouse development and consider the reasons why the requirement in ES cells do not reflect the embryo.

A rapid and efficient 2D/3D nuclear segmentation method for analysis of early mouse embryo and stem cell image data.

Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation) as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.

The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.

A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells.

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of 'self-renewal' and 'differentiation' genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and ß-catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

AID stabilizes stem-cell phenotype by removing epigenetic memory of pluripotency genes.

The activation-induced cytidine deaminase (AID; also known as AICDA) enzyme is required for somatic hypermutation and class switch recombination at the immunoglobulin locus. In germinal-centre B cells, AID is highly expressed, and has an inherent mutator activity that helps generate antibody diversity. However, AID may also regulate gene expression epigenetically by directly deaminating 5-methylcytosine in concert with base-excision repair to exchange cytosine. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells. However, different studies have suggested either a requirement or a lack of function for AID in promoting pluripotency in somatic nuclei after fusion with embryonic stem cells. Here we tested directly whether AID regulates epigenetic memory by comparing the relative ability of cells lacking AID to reprogram from a differentiated murine cell type to an induced pluripotent stem cell. We show that Aid-null cells are transiently hyper-responsive to the reprogramming process. Although they initiate expression of pluripotency genes, they fail to stabilize in the pluripotent state. The genome of Aid-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably upregulated, including many MYC target genes. Recent studies identified a late step of reprogramming associated with methylation status, and implicated a secondary set of pluripotency network components. AID regulates this late step, removing epigenetic memory to stabilize the pluripotent state.

Live imaging, identifying, and tracking single cells in complex populations in vivo and ex vivo.

Advances in optical imaging technologies combined with the use of genetically encoded fluorescent proteins have enabled the visualization of stem cells over extensive periods of time in vivo and ex vivo. The generation of genetically encoded fluorescent protein reporters that are fused with subcellularly localized proteins, such as human histone H2B, has made it possible to direct fluorescent protein reporters to specific subcellular structures and identify single cells in complex populations. This facilitates the visualization of cellular behaviors such as division, movement, and apoptosis at a single-cell resolution and, in principle, allows the prospective and retrospective tracking towards determining the lineage of each cell.

Correlations between the levels of Oct4 and Nanog as a signature for naïve pluripotency in mouse embryonic stem cells.

The pluripotent state is traditionally associated with large absolute levels of certain transcription factors such as Nanog and Oct4. Here, we present experimental observations using quantitative immunofluorescence that pluripotency in mouse embryonic stem cells (mESCs) is established by specific ratios between Oct4 and Nanog. When cells are grown in 2i conditions, they exhibit uniform levels of pluripotency and this is associated with a high correlation between the levels of Oct4 and Nanog in individual cells. The correlation is lost when cells differentiate. Our results suggest that the correlation between these two factors and the distribution of Oct4/Nanog ratios can be used as quantifiers to distinguish between three subpopulations in an mESC culture: pluripotent, lineage-primed, and differentiating cells. When we apply these quantifiers to cells with lower levels of Nanog or mutant for ß-Catenin or Tcf3, the results suggest that these cells exhibit higher probability of differentiation.

The structure of Wntch signalling and the resolution of transition states in development.

During development, the emergence of different cell fates and their patterning into tissues and organs requires spatio-temporal coordination that controls the relative number of different cell types. Genetic analyses in different systems have revealed that interactions between Wnt and Notch signalling play pervasive roles in these processes. While many of these interactions can be explained in terms of transcriptional cross-talk between the effectors of these pathways, some of them require a different explanation. Experiments in Drosophila, Xenopus and mouse have revealed that Notch plays an important role in the modulation of the transcriptional activity of ß-catenin (the main effector of Wnt signalling pathway, independently of its well characterized function as a membrane tethered transcription factor. These studies suggest that rather than two separate pathways, elements of Wnt and Notch signalling configure a single functional module, Wntch, that plays a key role in the resolution of cell fate decisions. Here we review the evidence for Wntch and present a current circuit view of the system, its control and its role in development with a special focus on stem cell populations.

Wnt-Notch signalling: an integrated mechanism regulating transitions between cell states.

The activity of Wnt and Notch signalling is central to many cell fate decisions during development and to the maintenance and differentiation of stem cell populations in homeostasis. While classical views refer to these pathways as independent signal transduction devices that co-operate in different systems, recent work has revealed intricate connections between their components. These observations suggest that rather than operating as two separate pathways, elements of Wnt and Notch signalling configure an integrated molecular device whose main function is to regulate transitions between cell states in development and homeostasis. Here, we propose a general framework for the structure and function of the interactions between these signalling systems that is focused on the notion of 'transition states', i.e. intermediates that arise during cell fate decision processes. These intermediates act as checkpoints in cell fate decision processes and are characterised by the mixed molecular identities of the states involved in these processes.

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila.

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of ß-catenin. Here, we show that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module.

Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye.

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.

Wingless modulates the ligand independent traffic of Notch through Dishevelled.

Here we investigate the structural and functional basis of the interactions between Notch and Wingless signalling in Drosophila. Using yeast-two-hybrid and pull-down assays we show that Notch can bind directly a form of Dishevelled that is stabilized upon Wingless signalling. Moreover, we show that the mechanism by which Wingless signalling is able to downregulate Notch is by promoting its ligand-independent traffic to a compartment where it is degraded and that this activity depends on Dishevelled.