Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images

NucleusJ: an ImageJ plugin for quantifying 3D images of inter- phase nuclei

ABSTRACT Summary: NucleusJ is a simple and user-friendly ImageJ plugin dedicated to the characterization of nuclear morphology and chromatin organization in 3D. Starting from image stacks, the nuclear boundary is delimited by combining the Otsu segmentation method with optimization of nuclear sphericity. Chromatin domains are segmented by partitioning the nucleus using a 3D watershed algorithm and by thresholding a contrast measure over the resulting regions. As output, NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for selftraining, together with data sets of nuclei with different nuclear organization. Availability: Data set of nuclei is available at https://www.gredclermont.fr/media/WorkDirectory.zip. NucleusJ is available at http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:nuclear_analysis_plugin:start

Study of the role of plant nuclear envelope and lamina-like components in nuclear and chromatin organisation using 3D imaging

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The fi aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphol- ogy and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with diff t nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromo- centres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC com- plex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes ad- vantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional NE. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes

Analysis of Spatial Point Patterns in Nuclear Biology

There is considerable interest in cell biology in determining whether, and to what extent, the spatial arrangement of nuclear objects affects nuclear function. A common approach to address this issue involves analyzing a collection of images produced using some form of fluorescence microscopy. We assume that these images have been successfully pre-processed and a spatial point pattern representation of the objects of interest within the nuclear boundary is available. Typically in these scenarios, the number of objects per nucleus is low, which has consequences on the ability of standard analysis procedures to demonstrate the existence of spatial preference in the pattern. There are broadly two common approaches to look for structure in these spatial point patterns. First a spatial point pattern for each image is analyzed individually, or second a simple normalization is performed and the patterns are aggregated. In this paper we demonstrate using synthetic spatial point patterns drawn from predefined point processes how difficult it is to distinguish a pattern from complete spatial randomness using these techniques and hence how easy it is to miss interesting spatial preferences in the arrangement of nuclear objects. The impact of this problem is also illustrated on data related to the configuration of PML nuclear bodies in mammalian fibroblast cells

Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos

Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased—thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization—and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation—the parthenote—and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research

Cloning in Cattle

In mammalian cell nuclei chromosome territories (CTs) occupy positions correlating with their gene-density and chromosome size. While this global radial order has been well documented, the question of whether a global neighborhood order is also maintained has remained a controversial matter. To answer this question I grew clones (of HeLa, HMEC and human diploid fibroblast cells) for up to 5 divisions (32 cells) and performed 3D FISH experiments to visualize the nuclear positions of 3 different CT pairs. Using different landmark-based registration approaches I assessed the similarity of CT arrangements in daughter cells and cousins. As expected from a symmetrical chromatid movement during mitotic anaphase and telophase, I was able to confirm previous findings of a pronounced similarity of CT arrangements between daughter cells. However, already after two cell cycles the neighborhood order in cousins was nearly completely lost. This loss indicates that a global neighborhood order is not maintained. Further, I could show in the present thesis that a gene density correlated distribution of CTs, which has already been shown in different cell types of various species appears to be independent of the cell cycle. Moreover I could provide evidence that the nuclear shape plays a major role in defining the extent of this gene-density correlated distribution, as nuclei of human, old world monkey and bovine fibroblasts showed an increased difference in the radial distribution of gene poor/dense CTs when their nuclei were artificially reshaped from a flat ellipsoid to a nearly spherical nucleus. The observation that a gene-density correlated distribution of CTs has been found in nuclei from birds to humans argues for a significant, yet undiscovered functional impact. So far CTs have been investigated mainly in cultured cells and to some extent in tissues, yet little is known about the origin and fate of CTs during early development. To gain insights into the very early organization of CTs in preimplantation embryos I have developed a fluorescence in situ hybridization (FISH) protocol, which enables the visualization of CTs in three dimensionally preserved embryos. Using this protocol I have investigated CTs of bovine chromosomes 19 and 20, representing the most gene-rich and gene-poor chromosomes, respectively. Equivalent to the distributions described in other species I could confirm a gene density related spatial CT arrangement in bovine fibroblasts and lymphocytes with CT 19 being localized more internally and CT 20 more peripherally. Importantly, I did not find a gene density related distribution of CTs 19 and 20 in early embryos up to the 8-cell stage. Only in embryos with more than 8 cells a significant difference in the distribution of both chromosomes became apparent that increased upon progression to the blastocyst stage. Since major genome activation in bovine embryos occurs during the 8- to 16-cell stage, my findings suggest an interrelation between higher order chromatin arrangements and transcriptional activation of the embryonic genome. Using another experimental set up I analyzed the topology of a developmentally regulated transgene utilizing bovine nuclear transfer (NT) embryos derived from fetal fibroblasts, which harbored a mouse Oct4/GFP reporter construct integrated at a single insertion site on bovine chromosome 13. I analyzed the intranuclear distribution of the transgene as well as its position in relation to its harboring chromosome in donor cell nuclei and day 2 NT embryos, where the transgene is still inactive as well as in day 4 NT embryos, where transgene expression starts, and day 7 NT embryos, where expression is highly increased. Compared to donor cell nuclei I found a more peripheral location of both BTA 13 CTs and the Oct4/GFP transgene in day 2, day 4 and day 7 NT embryos, although there was a trend of the transgene and both BTA 13 CTs to re-localize towards the nuclear interior from d2 to d7 embryos. Moreover, I found the transgene located at the surface of its harboring CT 13 in donor fibroblasts, whereas during preimplantation development of NT embryos it became increasingly internalized into the chromosome 13 territory, reaching a maximum in d7 NT embryos, i.e. at the developmental stage when its transcription levels are highest. These latter experiments show that the transfer of a somatic nucleus into a chromosome depleted oocyte triggers a large scale positional change of CTs 13 and of an Oct4/GFP transgene and indicate a redistribution of this developmentally regulated Oct4/GFP transgene during activation and upregulation in developing NT embryos