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Supplementary Figures S1 and S2 from A Mouse Model for Human Anal Cancer

The cultivation of cells forming three-dimensional structures like organoids holds great potential in different fields of life sciences and is gaining increasing interest with regards to clinical applications and personalised medicine. However, conventional hydrogels used as cell cultivation matrices (e.g. Matrigel®) contain animal-derived components in varying quantities, implicating low reproducibility of experiments and limited applicability for clinical use. Based on the strong need for developing novel, well defined, and animal-free hydrogels for 3D cell cultures, this study presents a comprehensive analysis of pancreas organoid cultivation in two synthetic hydrogels. Besides established visualisation techniques to monitor organoid formation and growth, confocal Raman microscopy was used for the first time to evaluate the gel matrices and organoid formation within the gels. The approach revealed so far not accessible information about material-cell interactions and the composition of the organoid lumen in a non-invasive and label-free manner. Confocal Raman microscopy thereby enabled a systematic characterisation of different hydrogels with respect to cell culture compatibility and allowed for the rational selection of a hydrogel formulation to serve as a synthetic and fully defined alternative to animal-derived cultivation matrices.

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/μm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angle towards the available water. Our observations reveal how the external hydrological environment regulates lateral root morphogenesis. Arabidopsis lateral root radial emergence is spatially constrained by the dual xylem poles in the main root. However, the asymmetric presence of water can modify the orientation of lateral roots at a very early stage.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

A widespread application of three-dimensional (3D) bioprinting in basic and translational research requires the accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithographic (SL) printer was developed. This SL bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a highly parallelized tissue production.To showcase the capability of the bioprinter, we produced constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel. A thorough characterization of different GelMA/PEGDA ratios revealed that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirmed that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.

Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future. Light sheet fluorescence microscopy (LSFM) is a technique that uses a thin sheet of light for illumination, allowing optical sectioning of the sample. In this Primer, Stelzer et al. outline the fundamental concepts behind LSFM, discuss the different experimental set-ups for light sheet microscopes and detail steps for processing LSFM images. The Primer also describes the range of applications for this technique across the biological sciences and concludes by discussing advances for enhancing imaging depth and resolution.

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.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44−/− mice but not Cd14−/− mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Organoids are morphologically heterogeneous three-dimensional cell culture systems. To understand the cell organisation principles of their morphogenesis, we imaged hundreds of pancreas and liver organoids in parallel using light sheet and bright field microscopy for up to seven days. We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation and degeneration, and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright field macroscale analyses of entire cultures. Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying core regulatory principles of organoid morphogenesis.Graphical AbstractCreated with BioRender.com.

This document presents the first version of the Climate Assessment Report (CAR), which will be updated in 2021, to summarize the added value of Lakes_cci products and the activities of the Climate Research Group (CRG). Overall, the CAR first introduces the products generated by Lakes_cci. A second section on initial data exploration at global scale is also presented along with sample associated methodological steps. The specific activities from the CRG on five pre-defined use cases is then presented for addressing a variety of scientific questions. The CAR concludes confronting the progress made towards meeting user requirements and also includes main outcomes from the Lake_cci user workshop. This is followed by an updated list of publications, a summary of outreach activities and key conclusions.

Nature chemical biology 16, 1-19 (2020). doi:10.1038/s41589-020-0600-3, The p53 homolog TAp63$α$ is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63$α$ is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63$α$ phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63$α$ substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one., Published by Nature Publishing Group, Basingstoke

Phosphatidic acid (PA) lipids have a dual role as building blocks for membrane biogenesis and as active signaling molecules. This study establishes the molecular details of selective PA recognition by the transcriptional regulator Opi1 from baker’s yeast., A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for selective binding of PA over phosphatidylserine (PS). The insertion of the AH into the membrane core renders Opi1 sensitive to the lipid acyl chain composition and provides a means to adjust membrane biogenesis. By rational design of the AH, we tune the membrane-binding properties of Opi1 and control its responsiveness in vivo. Using extensive molecular dynamics simulations, we identify two PA-selective three-finger grips that tightly bind the PA phosphate headgroup while interacting less intimately with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.

Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system., The GABAergic and peptidergic neuron RIS mediates sleep in Caenorhabditis elegans. The authors demonstrated here that RIS also functions as a locomotion stop neuron. Its optogenetic stimulation caused acute and persistent inhibition of locomotion, and brief intrinsic RIS activity preceded slowing.

International audience; The coastal erosion is defined as the loss or displacement of land along the coastline due to the action of rain, ice and atmospheric and marine forcing. In Europe, about 25% of the European coastline for which data is available is currently retreating and erosion up to 20-30 m when severe storm occurs are frequent. In the context of climate change and sea level rise, the coastal erosion issue will be even more relevant in the future, as both the frequency and strength of storm events are likely to increase causing billions of Euros of damages. Dealing with coastal erosion implies to provide highly precise (sub-metric) geo-located information about shorelines on a yearly to decadal timescale. For this reason, the classical approach for coastal erosion assessment has been based on the analysis of historical aerial orthophotos (enabling the extraction of longterm trends), along with GPS field observations (short-term beach and shoreline changes at the timescale of storm events). These past 20 years, the use of new technologies for coastal change and shoreline monitoring has significantly increased (airborne lidar topographic surveys, photogrammetry, in situ laser scanning) but their definitive adoption still depends on their cost to effectiveness ratio. In the meanwhile, satellite remote sensing has shown to be adequate and relatively cost-effective for the extraction and monitoring of shoreline change over sandy coasts, and other relevant indicators of coastal erosion (beach and cliff morphological patterns, nearshore bathymetry, tidal flat change in semi-enclosed bays,…). In the framework of the ESA Coastal Erosion Project, the future “Space for Shore” service intends to unravel the remaining technical issues and to provide a large European end user community spread over 5 countries with prototyped products, that are based on the Copernicus Sentinel-1 and Sentinel-2 missions and, to a certain extent, on Third Party Missions. The challenge definitely resides in the adequation between EO-data ground resolution, satellite product accuracy and the rate of coastal erosion and shoreline change, collectively targeting the retrieval of major coastal indicators designed to support decision-making. This communication proposes an overview of the first results obtained from the Proof-of-Concept activities run this year, accordingly to the set of products required by European coastal managers in charge of shoreline management. A soundful validation of these prototyped products is also performed assessing satellite real capability to meet end user requirements in terms mainly of measurement accuracy and temporal revisit. Finally, in conclusion, benefits for the coastal community are addressed along with some perspectives for wide service deployment over Europe.

During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first cell fate decision, cells become either part of an outer trophectoderm or part of the inner cell mass. Subsequently, the inner cell mass (ICM) segregates into an embryonic and an extraembryonic lineage, giving rise to the epiblast and the primitive endoderm, respectively. Inner cell mass organoids represent an experimental model system for preimplantation development, mimicking the second cell fate decision taking place inin vivomouse embryos. In a previous study, the spatial pattern of the different cell lineage types was investigated. The study revealed that cells of the same fate tend to cluster stronger than expected for purely random cell fate decisions. Three major processes are hypothesised to contribute to the final cell fate arrangements at the mid and late blastocysts or 24 h old and 48 h old ICM organoids, respectively: 1) intra- and intercellular chemical signalling; 2) a cell sorting process; 3) cell proliferation. In order to quantify the influence of cell proliferation on the emergence of the observed cell lineage type clustering behaviour, we developed an agent-based model. Hereby, cells are mechanically interacting with direct neighbours, and exert adhesion and repulsion forces. The model was applied to compare several current assumptions of how inner cell mass neighbourhood structures are generated. We tested how different assumptions regarding cell fate switches affect the observed neighbourhood relationships. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. The model further shows that the observed neighbourhood structures can emerge due to cell fate heredity during cell division and allows the inference of a time point for the cell fate decision.STATEMENT OF SIGNIFICANCECell fate decisions in early embryogenesis have been considered random events, causing a random cell fate distribution. Using an agent-based mathematical model, fitted to ICM organoid data, we show that the assumed random distribution of cell fates occurs only for a short time interval, as cell fate heredity and cell division quickly lead to spatial cell fate clustering. Our results show that neighbourhood clustering can emerge without specific neighbourhood interactions affecting the cell fate decision. The approach indicates four consecutive phases of early development: 1) co-expression of cell fate markers, 2) cell fate decision, 3) division and local cell fate clustering, and 4) phase separation, whereby only the phases 1-3 occur in ICM organoids during the first 24h of growth.

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63α is the key regulator of genome integrity in oocytes. After DNA damage TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kineticsin vitrowith peptides and full length protein we show that TAp63α phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of theTc-foxQ2forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types ofTc-foxQ2positive neural progenitor cells based on differential co-expression withTc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rxandTc-fez1. An enhancer trap line built by genome editing markedTc-foxQ2positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, inTc-foxQ2RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishesfoxQ2as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.Summary statementAn ancestral neuroendocrine center contributes to the evolution of the central complex.foxQ2is a gene required for the development of midline structures of the insect brain, which distinguish protocerebrum from segmental ganglia.

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angle towards the available water. Our observations reveal how the external hydrological environment regulates lateral root morphogenesis.

Oceanic shelf sea fronts have significant effects on local dynamics, ecology and climate. An assessment of the impact of climate change on frontal positions and frontal gradients requires reliable reference data and the possibility to monitor oceanic fronts. Therefore, the development of algorithms which automatically detect frontal positions from Earth Observation (EO) data is an important tool to analyse long EO time series, i.e. to process big data volumes. The development of GRADHIST was driven by the need to generate a climatology for North Sea fronts. GRADHIST is a new algorithm for the detection and mapping of oceanic fronts, which is based on a combination and refinement of the gradient algorithm of Canny (1986) and the histogram algorithm of Cayula and Cornillon (1992). GRADHIST preserves the main principles of both algorithms and can be applied to various ocean parameters as well as to different sensors with very little effort. GRADHIST was validated and tested using both synthetic and real data and applied to sea surface temperature and ocean colour parameters retrieved from satellite data; i.e. data from MODIS (Moderate Resolution Imaging Spectroradiometer), MERIS (MEdium Resolution Imaging Spectrometer), AVHRR (Advanced Very High Resolution Radiometer) and AATSR (Advanced Along-Track Scanning Radiometer). Selected results and statistical analysis of a new North Sea climatology for oceanic fronts are presented and discussed.

In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.

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., Summary: The phases of spheroid formation resemble different stages of cell contact formation. This facilitates studying the temporal contribution of molecules in this process.

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.

The optical sectioning capability is fundamental for three-dimensional imaging. One of the very few microscopes, which claim this property is light sheet-based fluorescence microscopy (LSFM). In general, FM provides a high contrast, since only specifically labelled cellular components are observed while all other structures remain “dark”. However: 1) Excitation light degrades endogenous organic compounds and bleaches fluorophores. 2) Only a finite number of excitable fluorophores is available, which limits the quantity of collectable emitted photons. 3) Organisms are adapted to the solar flux of 1.4 kW/m2. LSFM decouples the excitation and emission light pathways. The optical axis of the illumination objective lens is aligned with the focal plane of the perpendicularly arranged detection objective lens. By design, only the fluorophores around the focal plane are excited for each acquired two-dimensional image. Hence, each fluorophore is exposed only once to the illumination light when a three-dimensional image is recorded. The significance of the illumination-based optical sectioning property is that the viability and the fluorescence signal of a living specimen are retained while millions of images are recorded for days or even weeks. Further benefits: (i) a good axial resolution, (ii) imaging along multiple directions, (iii) deeper tissue penetration due to the low numerical aperture of the illumination objective lens, (iv) a high signal-to-noise ratio, (v) an unrestricted compatibility with fluorescent dyes and proteins, (vi) reduced fluorophore bleaching and (vii) photo-toxicity at almost any scale, (viii) millions of pixels are recorded in parallel and (ix) a dramatically improved viability of the specimen.