Structure tensor based analysis of cells and nuclei organization in tissues

International audienceMotivation: Extracting geometrical information from large 2D or 3D biomedical images is important to better understand fundamental phenomena such as morphogenesis. We address the problem of automatically analyzing spatial organization of cells or nuclei in 2D or 3D images of tissues. This problem is challenging due to the usually low quality of microscopy images as well as their typically large sizes. Results: The structure tensor is a simple and robust descriptor that was developed to analyze textures orientation. Contrarily to segmentation methods which rely on an object based modelling of images, the structure tensor views the sample at a macroscopic scale, like a continuum. We propose an original theoretical analysis of this tool and show that it allows quantifying two important features of nuclei in tissues: their privileged orientation as well as the ratio between the length of their main axes. A quantitative evaluation of the method is provided for synthetic and real 2D and 3D images. As an application, we analyze the nuclei orientation and anisotropy on multicellular tumor spheroids cryosections. This analysis reveals that cells are elongated in a privileged direction that is parallel to the boundary of the spheroid. Availability: Source codes are available at http://www.math.univ-toulouse.fr/~weiss

Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation <em>in vivo</em>

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome reorganisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGF beta). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation

Mental Fatigue Negatively Influences Manual Dexterity and Anticipation Timing but not Repeated High-intensity Exercise Performance in Trained Adults

This study examined the impact of a period of mental fatigue on manual dexterity, anticipation timing and repeated high intensity exercise performance. Using a randomised, repeated measures experimental design, eight physically trained adults (mean age = 24.8 ± 4.1 years) undertook a 40 minute vigilance task to elicit mental fatigue or a control condition followed by four repeated Wingate anaerobic performance tests. Pre, post fatigue/control and post each Wingate test, manual dexterity (Seconds), coincidence anticipation (absolute error) were assessed. A series of two (condition) by six (time) ways repeated measures ANOVAs indicated a significant condition by time interactions for manual dexterity time (p = 0.021) and absolute error (p = 0.028). Manual dexterity and coincidence anticipation were significantly poorer post mental fatigue compared with control. There were no significant differences in mean power between conditions or across trials (all p &gt; 0.05). Publisher statement: This is an Accepted Manuscript of an article published by Taylor &amp; Francis in Research in Sports Medicine: An International Journal on 29th January 2015, available online: http://www.tandfonline.com/doi/full/10.1080/15438627.2014.97581

Cyclin D1 promotes neurogenesis in the developing spinal cord in a cell cycle-independent manner

Neural stem and progenitor cells undergo an important transition from proliferation to differentiation in the G1 phase of the cell cycle. The mechanisms coordinating this transition are incompletely understood. Cyclin D proteins promote proliferation in G1 and typically are down-regulated before differentiation. Here we show that motoneuron progenitors in the embryonic spinal cord persistently express Cyclin D1 during the initial phase of differentiation, while down-regulating Cyclin D2. Loss-of-function and gain-offunction experiments indicate that Cyclin D1 (but not D2) promotes neurogenesis in vivo, a role that can be dissociated from its cell cycle function. Moreover, reexpression of Cyclin D1 can restore neurogenic capacity to D2-expressing glial-restricted progenitors. The neurogenic function of Cyclin D1 appears to be mediated, directly or indirectly, by Hes6, a proneurogenic basic helic-loop-helix transcription factor. These data identify a cell cycle-independent function for Cyclin D1 in promoting neuronal differentiation, along with a potential genetic pathway through which this function is exerted

Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord

<p>Abstract</p> <p>Background</p> <p>During the development of the nervous system, neural progenitor cells can either stay in the pool of proliferating undifferentiated cells or exit the cell cycle and differentiate. Two main factors will determine the fate of a neural progenitor cell: its position within the neuroepithelium and the time at which the cell initiates differentiation. In this paper we investigated the importance of the timing of cell cycle exit on cell-fate decision by forcing neural progenitors to cycle and studying the consequences on specification and differentiation programs.</p> <p>Results</p> <p>As a model, we chose the spinal progenitors of motor neurons (pMNs), which switch cell-fate from motor neurons to oligodendrocytes with time. To keep pMNs in the cell cycle, we forced the expression of G1-phase regulators, the D-type cyclins. We observed that keeping neural progenitor cells cycling is not sufficient to retain them in the progenitor domain (ventricular zone); transgenic cells instead migrate to the differentiating field (mantle zone) regardless of cell cycle exit. Cycling cells located in the mantle zone do not retain markers of neural progenitor cells such as Sox2 or Olig2 but upregulate transcription factors involved in motor neuron specification, including MNR2 and Islet1/2. These cycling cells also progress through neuronal differentiation to axonal extension. We also observed mitotic cells displaying all the features of differentiating motor neurons, including axonal projection via the ventral root. However, the rapid decrease observed in the proliferation rate of the transgenic motor neuron population suggests that they undergo only a limited number of divisions. Finally, quantification of the incidence of the phenotype in young and more mature neuroepithelium has allowed us to propose that once the transcriptional program assigning neural progenitor cells to a subtype of neurons is set up, transgenic cells progress in their program of differentiation regardless of cell cycle exit.</p> <p>Conclusion</p> <p>Our findings indicate that maintaining neural progenitor cells in proliferation is insufficient to prevent differentiation or alter cell-fate choice. Furthermore, our results indicate that the programs of neuronal specification and differentiation are controlled independently of cell cycle exit.</p

Deep and Clear Optical Imaging of Thick Inhomogeneous Samples

Inhomogeneity in thick biological specimens results in poor imaging by light microscopy, which deteriorates as the focal plane moves deeper into the specimen. Here, we have combined selective plane illumination microscopy (SPIM) with wavefront sensor adaptive optics (wao). Our waoSPIM is based on a direct wavefront measure using a Hartmann-Shack wavefront sensor and fluorescent beads as point source emitters. We demonstrate the use of this waoSPIM method to correct distortions in three-dimensional biological imaging and to improve the quality of images from deep within thick inhomogeneous samples

Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy

<p>Abstract</p> <p>Background</p> <p>Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.</p> <p>Results</p> <p>Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.</p> <p>Conclusions</p> <p>As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.</p

Learning the cell cycle with a game: Virtual experiments in cell biology

Cell Cycle Learn (CCL) is a learning game designed for undergraduate students in Biology to learn common knowledge about the cell-division cycle along with practical skills related with setting up an experiment and the scientific method in general. In CCL, learners are guided through the process of formulating hypotheses, conducting virtual experiments and analysing the results in order to validate or invalidate the hypotheses. The game has been designed in the University of Toulouse and introduced last year as part of the curriculum of a cellular biology class. This paper presents early results of an evaluation of the game enabled by questionnaires filled by the participants and game data collected during the training sessions. The results demonstrate with examples that both types of data can be used to assess the game's utility

Genetic variability of transcript abundance in pig peri-mortem skeletal muscle: eQTL localized genes involved in stress response, cell death, muscle disorders and metabolism

<p>Abstract</p> <p>Background</p> <p>The genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering.</p> <p>Results</p> <p>QTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative <it>cis</it>-drivers of gene expression programs in skeletal muscle as <it>COQ4 </it>(SSC1), <it>LOC100513192 </it>(SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests <it>cis</it>-causing genetic polymorphims affecting gene expression levels, with (e.g. <it>COQ4</it>) or without (e.g. <it>LOC100513192</it>) potential pleiotropic effects that affect the expression of other genes (cluster of <it>trans</it>-eQTLs).</p> <p>Conclusion</p> <p>Genetic analysis of transcription levels revealed dependence among molecular phenotypes as being affected by variation at the same loci. We observed the genetic variation of molecular phenotypes in a specific situation of cellular stress thus contributing to a better description of muscle physiologic response. In turn, this suggests that large amounts of genetic variation, mediated through transcriptional networks, can drive transient cell response phenotypes and contribute to organismal adaptative potential.</p

Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

Abstract Background MultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D. Methods Cell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters. Results We describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage. Conclusion Our data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models. </jats:sec