Modelling polarity-driven laminar patterns in bilayer tissues with mixed signalling mechanisms

Recent advances in high-resolution experimental methods have highlighted the significance of cell signal pathway crosstalk and localised signalling activity in the development and disease of numerous biological systems. The investigation of multiple signal pathways often introduces different methods of cell-cell communication, i.e. contact-based or diffusive signalling, which generates both a spatial and temporal dependence on cell behaviours. Motivated by cellular mechanisms that control cell-fate decisions in developing bilayer tissues, we use dynamical systems coupled with multilayer graphs to analyse the role of signalling polarity and pathway crosstalk in fine-grain pattern formation of protein activity. Specifically, we study how multilayer graph edge structures and weights influence the layer-wise (laminar) patterning of cells in bilayer structures, which are commonly found in glandular tissues. We present sufficient conditions for existence, uniqueness and instability of homogeneous cell states in the large-scale spatially discrete dynamical system. Using methods of pattern templating by graph partitioning to generate quotient systems in combination with concepts from monotone dynamical systems, we exploit the extensive dimensionality reduction to provide existence conditions for the polarity required to induce fine-grain laminar patterns with multiple spatially dependent intracellular components. We then explore the spectral links between the quotient and large-scale dynamical systems to extend the laminar patterning criteria from existence to convergence for sufficiently large amounts of cellular polarity in the large-scale dynamical system, independent of spatial dimension and number of cells in the tissue

A divergent canonical WNT-signaling pathway regulates microtubule dynamics: Dishevelled signals locally to stabilize microtubules

Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability

Modelling polarity-driven laminar patterns in bilayer tissues with mixed signalling mechanisms

Recent advances in high-resolution experimental methods have highlighted the significance of cell signal pathway crosstalk and localised signalling activity in the development and disease of numerous biological systems. The investigation of multiple signal pathways often introduces different methods of cell-cell communication, i.e. contact-based or diffusive signalling, which generates both a spatial and temporal dependence on cell behaviours. Motivated by cellular mechanisms that control cell-fate decisions in developing bilayer tissues, we use dynamical systems coupled with multilayer graphs to analyse the role of signalling polarity and pathway crosstalk in fine-grain pattern formation of protein activity. Specifically, we study how multilayer graph edge structures and weights influence the layer-wise (laminar) patterning of cells in bilayer structures, which are commonly found in glandular tissues. We present sufficient conditions for existence, uniqueness and instability of homogeneous cell states in the large-scale spatially discrete dynamical system. Using methods of pattern templating by graph partitioning to generate quotient systems, in combination with concepts from monotone dynamical systems, we exploit the extensive dimensionality reduction to provide existence conditions for the polarity required to induce fine-grain laminar patterns with multiple spatially dependent intracellular components. We then explore the spectral links between the quotient and large-scale dynamical systems to extend the laminar patterning criteria from existence to convergence for sufficiently large amounts of cellular polarity in the large-scale dynamical system, independent of spatial dimension and number of cells in the tissue

Identifying subpopulations in multicellular systems by quantitative chemical imaging using label-free hyperspectral CARS microscopy

Quantitative hyperspectral coherent Raman scattering microscopy merges imaging with spectroscopy and utilises quantitative data analysis algorithms to extract physically meaningful chemical components, spectrally and spatially-resolved, with sub-cellular resolution. This label-free non-invasive method has the potential to significantly advance our understanding of the complexity of living multicellular systems. Here, we have applied an in-house developed hyperspectral coherent anti-Stokes Raman scattering (CARS) microscope, combined with a quantitative data analysis pipeline, to imaging living mouse liver organoids as well as fixed mouse brain tissue sections xenografted with glioblastoma cells. We show that the method is capable of discriminating different cellular sub-populations, on the basis of their chemical content which is obtained from an unsupervised analysis, i.e. without prior knowledge. Specifically, in the organoids, we identify sub-populations of cells at different phases in the cell cycle, while in the brain tissue, we distinguish normal tissue from cancer cells, and, notably, tumours derived from transplanted cancer stem cells versus non-stem glioblastoma cells. The ability of the method to identify different sub-populations was validated by correlative fluorescence microscopy using fluorescent protein markers. These examples expand the application portfolio of quantitative chemical imaging by hyperspectral CARS microscopy to multicellular systems of significant biomedical relevance, pointing the way to new opportunities in non-invasive disease diagnostics

Developmentally regulated Tcf7l2 splice variants mediate transcriptional repressor functions during eye formation

Tcf7l2 mediates Wnt/b-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/b-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2 alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway

A functional connectome: regulation of Wnt/TCF-dependent transcription by pairs of pathway activators.

BACKGROUND: Wnt/β-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between 'core pathway' members, to the induction of nuclear transcription from genes flanked by β-catenin/TCF transcription factor binding sites. Wnt/β-catenin signaling is also regulated by a much larger set of 'non-core regulators'. However the relationship between 'non-core regulators' is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues. METHODS: Mammalian cells engineered to have a partially-active level of Wnt/β-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP). RESULTS: 141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, 'core pathway' components (Eg. β-catenin, GSK-3, Dsh) were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists. CONCLUSIONS: The 'functional connectome' identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment

Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids

The development of in vitro culture systems quantitatively and qualitatively recapitulating normal breast biology is key to the understanding of mammary gland biology. Current three-dimensional mammary culture systems have not demonstrated concurrent proliferation and functional differentiation ex vivo in any system for longer than 2 weeks. Here, we identify conditions including Neuregulin1 and R-spondin 1, allowing maintenance and expansion of mammary organoids for 2.5 months in culture. The organoids comprise distinct basal and luminal compartments complete with functional steroid receptors and stem/progenitor cells able to reconstitute a complete mammary gland in vivo. Alternative conditions are also described that promote enrichment of basal cells organized into multiple layers surrounding a keratinous core, reminiscent of structures observed in MMTV-Wnt1 tumours. These conditions comprise a unique tool that should further understanding of normal mammary gland development, the molecular mechanism of hormone action and signalling events whose deregulation leads to breast tumourigenesis

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model

Functional and molecular characterisation of mammary side population cells

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2–0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology

Large Scale Benchmark of Materials Design Methods

Lack of rigorous reproducibility and validation are major hurdles for scientific development across many fields. Materials science in particular encompasses a variety of experimental and theoretical approaches that require careful benchmarking. Leaderboard efforts have been developed previously to mitigate these issues. However, a comprehensive comparison and benchmarking on an integrated platform with multiple data modalities with both perfect and defect materials data is still lacking. This work introduces JARVIS-Leaderboard, an open-source and community-driven platform that facilitates benchmarking and enhances reproducibility. The platform allows users to set up benchmarks with custom tasks and enables contributions in the form of dataset, code, and meta-data submissions. We cover the following materials design categories: Artificial Intelligence (AI), Electronic Structure (ES), Force-fields (FF), Quantum Computation (QC) and Experiments (EXP). For AI, we cover several types of input data, including atomic structures, atomistic images, spectra, and text. For ES, we consider multiple ES approaches, software packages, pseudopotentials, materials, and properties, comparing results to experiment. For FF, we compare multiple approaches for material property predictions. For QC, we benchmark Hamiltonian simulations using various quantum algorithms and circuits. Finally, for experiments, we use the inter-laboratory approach to establish benchmarks. There are 1281 contributions to 274 benchmarks using 152 methods with more than 8 million data-points, and the leaderboard is continuously expanding. The JARVIS-Leaderboard is available at the website: https://pages.nist.gov/jarvis_leaderboar