67 CORRELATION OF ARCHITECTURAL AND FUNCTIONAL REPROGRAMMING OF NUCLEI DURING EMBRYONIC GENOME ACTIVATION OF EARLY BOVINE EMBRYOS GENERATED BY IVF AND SCNT

2014 ◽  
Vol 26 (1) ◽  
pp. 147
Author(s):  
J. Popken ◽  
D. Koehler ◽  
A. Brero ◽  
A. Wuensch ◽  
T. Thormeyer ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Nuclear Architecture ◽  
Nuclear Space ◽  
Structured Illumination ◽  
Bovine Embryos ◽  
Structured Illumination Microscopy ◽  
Nuclear Compartment ◽  
Nuclear Interior ◽  
Embryonic Genome ◽  
Genome Activation

Development of mammalian pre-implantation embryos provides an excellent model to explore interactions of nuclear organisation and nuclear functions. Based on light optical sectioning with confocal laser scanning microscopy and structured illumination microscopy, we performed a quantitative three-dimensional image analysis of nuclei in early bovine embryos generated by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) of bovine fibroblast nuclei. The same sequence of changes was observed in nuclei of both IVF and SCNT embryos during embryonic genome activation (EGA) is that typically achieved in embryos between 8 and 16 cells. In both pre-EGA IVF and SCNT embryos, chromosome territories (CT) were assembled as spatially distinct entities at the nuclear periphery, whereas the nuclear interior was typically occupied by a mostly chromatin free lacuna enriched with splicing factors. Detection of H3K4m3 demonstrates the presence of transcriptionally competent chromatin before EGA, which was correlated with large-scale movements of CT into the nuclear interior and a several-fold decrease of nuclear volumes. Post-EGA nuclei are characterised by a conventional nuclear architecture with chromatin distributed throughout the nuclear space, heterochromatin enriched with histone markers for transcriptionally silent chromatin beneath the nuclear lamina and around nucleoli, as well as heterochromatin clusters and chromocenters throughout the nuclear interior. Pre- and post-EGA nuclei were recorded with the superior resolution of structured illumination microscopy to allow a quantitative analysis of the nuclear topography of H3K4me3 and RNAP II signals. These signals were highly significantly enriched in the perichromatin region (PR) surrounding the compact, transcriptionally silent interior of megabase-sized chromatin domains, which form the basic structural units of CT. The PR is in direct contact with interchromatin compartment (IC) channels starting at nuclear pores, permeating the nuclear space and harboring nuclear bodies in IC lacunas. Our findings support a model for the functional nuclear architecture based on spatially distinct, but co-aligned three-dimensional networks of an active and an inactive nuclear compartment. The active nuclear compartment is built up from the structurally and functionally interacting IC and PR, whereas the inactive nuclear compartment consists of the compact, transcriptionally silent core of chromatin domain clusters. This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

scholarly journals Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

scholarly journals Chromatin remodeling in bovine embryos indicates species-specific regulation of genome activation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michelle M. Halstead ◽  
Xin Ma ◽  
Chuan Zhou ◽  
Richard M. Schultz ◽  
Pablo J. Ross
Keyword(s):  
Preimplantation Development ◽  
Open Chromatin ◽  
Bovine Embryos ◽  
Embryonic Genome ◽  
Epigenetic Remodeling ◽  
Profile Changes ◽  
Specific Regulation ◽  
Species Specific ◽  
Genome Activation ◽  
Cross Species Comparison

Abstract The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice.

69 GLOBAL H3k9ac AND H3k27me3 EXPRESSION IN BLASTOMERES FROM 8- TO 16-CELL STAGE BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 148
Author(s):  
C. S. Oliveira ◽  
N. Z. Saraiva ◽  
L. Z. Oliveira ◽  
R. V. Serapião ◽  
M. R. de Lima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Histone Modifications ◽  
Pearson Correlation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Group A ◽  
Genome Activation

Embryonic genome activation is a crucial step in early embryo development, and is accompanied by a dramatic change in the epigenetic profile of blastomeres. Histone modifications related to euchromatin and heterochromatin can be important parameters to infer developmental competence, as they are affected by manipulation and environmental stress conditions. The aim of this study was to characterise permissive (H3k9ac) and repressive (H3k27me3) histone modifications during the embryonic genome activation cell cycle in bovine embryos, regarding correlation between those marks and variance among blastomeres. For that, bovine embryos were produced by IVF and cultured in SOF medium supplemented with 5 mg mL–1 of BSA and 2.5% FCS in 5% O2 in an air atmosphere for 5 days (70 h after IVF). The 8 to 16 cell embryos were fixed in 4% paraformaldehyde and submitted to H3k9ac and H3k27me3 immunofluorescence assay (mouse anti-H3K9ac monoclonal antibody, 1 : 200; Sigma; rabbit anti-H3k27me3 monoclonal antibody, 1 : 200; Upstate, Charlottesville, VA, USA). Nuclei were counterstained with Hoechst 33342. Images of each embryo were captured (AxioCam, Carl Zeiss, São Paulo, Brazil) and measured for nuclear fluorescence intensity in each blastomere using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). Mean levels were compared using the Mann-Whitney test and variances were compared using F-test (SAS 9.1, SAS Institute Inc., Cary, NC, USA; P = 0.05). We evaluated 2 replicates and 12 embryos during the transition from the 8 to 16 cell stages, totaling 169 blastomeres. Global H3k27me3 levels varied accordingly to H3k9ac levels, as indicated by a high Pearson correlation coefficient (r = 0.913). Levels of each blastomere were normalized to the lowest level obtained within each embryo. Some embryos displayed a high variation between blastomeres, and, for further analysis, we divided the embryos into groups: group A for embryos that presented similar H3k9ac levels between blastomeres (8 embryos, 66%), and group B for embryos that exhibited higher heterogeneity between blastomeres (at least 2 blastomeres presenting a 2-fold increase compared to the lowest blastomere; 4 embryos, 33%). Mean H3k9ac and H3k27me3 normalized levels were lower for group A [H3k9ac: 1.35 ± 0.29 (A), 1.94 ± 1.02* (B); H3k27me3: 1.33 ± 0.24 (A), 1.99 ± 0.77 (B)], and group A displayed lower variance values (H3k9ac: 0.07 (A), 1.05* (B); H3k27me3: 0.06 (A), 0.60 (B)]. Within each embryo, blastomeres were sorted in ascending order for H3k9ac level (1 to 16), and compared between groups A and B. We detected that mean levels differed (P < 0.05) between groups from blastomere 9 to 16 for H3k9ac and 10 to 16 for H3k27me3. Therefore, in 8- to 16-cell stage embryos, the H3k27me3 repressive mark is highly correlated with the H3k9ac permissive mark. Also, our results describe the presence of 2 distinguishable populations of bovine embryos at this stage, considering their epigenetic status. One population presented similar levels of repressive and permissive marks among blastomeres, whereas the second one displayed a remarkable variation among their blastomeres. This observation should be further studied, as it might reflect distinct cleavage pattern embryos and blastomere competence. The authors acknowledge FAPESP, FAPERJ and CNPq for financial support.

scholarly journals A novel workflow for three-dimensional analysis of tumour cell migration

2020 ◽  
Vol 10 (2) ◽  
pp. 20190070 ◽  
Author(s):  
Sophie Ketchen ◽  
Arndt Rohwedder ◽  
Sabine Knipp ◽  
Filomena Esteves ◽  
Nina Struve ◽  
...  
Keyword(s):  
Dimensional Analysis ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tumour Spheroid ◽  
3D Data ◽  
Biological Studies ◽  
Research Findings ◽  
Imagej Plugin

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal–amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

scholarly journals High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

Nanophotonics ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 143-148
Author(s):  
Adrien Descloux ◽  
Marcel Müller ◽  
Vytautas Navikas ◽  
Andreas Markwirth ◽  
Robin van den Eynde ◽  
...  
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
Optical Element ◽  
Spatial Light Modulators ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Image Stack ◽  
Light Modulators ◽  
3D Information

AbstractSuper-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high-speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.

The role of RNA-polymerase II transcription in embryonic nucleologenesis by bovine embryos

Biologia ◽  
2010 ◽  
Vol 65 (3) ◽  
Author(s):  
Mária Kovalská ◽  
Ida Petrovičová ◽  
František Strejček ◽  
Marian Adamkov ◽  
Erika Halašová ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Control Group ◽  
Bovine Embryos ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Autoradiographic Labeling ◽  
Genome Activation

AbstractThe early stages of embryonic development are maternally driven. As development proceeds, maternally inherited informational molecules decay, and embryogenesis becomes dependent on de novo synthesized RNAs of embryonic genome. The aim of the present study is to investigate the role of de novo transcription in the development of embryos during embryonic genome activation. Autoradiography for detection of transcriptional activity and transmission electron microscopy were applied in in vitro produced bovine embryos cultured to the late 8-cell stage with or without (control group) α-amanitin, specific inhibitor of RNA-polymerases II and III transcription. The α-amanitin (AA) groups presented three sets of embryos cultivated with AA in different time intervals (6, 9 and 12 h). In control group, nucleoplasm and nucleolar structures displayed strong autoradiographic labeling and showed initial development of fibrillo-granular nucleoli. In α-amanitin groups, lack of autoradiographic labeling and disintegrated nucleolus precursor bodies (NPBs) stage were observed. Inhibition of RNA polymerase II (RNA pol II) already in the early phases of embryonic genome activation has detrimental effect on nucleolar formation and embryo survival, what was shown for the first time.

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