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The transition from local to global patterns governs the differentiation of mouse blastocysts
(2020)
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.
In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.
Spheroids resemble features of tissues and serve as model systems to study cell–cell and cell–ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue integrity. Tissue integrity involves the cellular processes adhesion formation, adhesion reinforcement, rearrangement as well as proliferation. They maintain the structure and function of tissues and, upon dysregulation, contribute to malignancy. We investigated spheroid formation dynamics in cell lines of different metastatic potential. We dissected spheroid formation into phases of aggregation, compaction and growth to identify the respective contributions of E-cadherin, actin, microtubules and FAK. E-cadherin, actin and microtubules drive the first two phases. Microtubules and FAK are involved in the proliferation phase. FAK activity correlates with the metastatic potential of the cells. A robust computational model based on a very large number of experiments reveals the temporal resolution of cell adhesion. Our results provide novel hypotheses to unveil the general mechanisms that contribute to tissue integrity.
Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid’s size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 105 to 1 × 106 cells/mm3. Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.
Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.
Endogenous AJAP1 associates with the cytoskeleton and attenuates angiogenesis in endothelial cells
(2017)
The adherens junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJAP1 is associated with alterations in cell migration, invasion, increased tumor growth, and tumor vascularization, suggesting AJAP1 as a putative tumor suppressor. We show that AJAP1 attenuates sprouting angiogenesis by reducing endothelial migration and invasion capacities. Further, we show for the first time that endogenous AJAP1 is associated with the microtubule cytoskeleton. This linkage is independent from cell confluency and stable during angiogenic sprouting in vitro. Our work suggests that AJAP1 is a putative negative regulator of angiogenesis, reducing cell migration and invasion by interfering with the microtubule network. Based on our results and those of other authors, we suggest AJAP1 as a novel tumor suppressor and diagnostic marker.
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue
(2017)
The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of T. castaneum adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.
Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells
(2013)
Cadherins, Ca2+-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca2+-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca2+-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca2+-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.